Synthesis of naturally occcuring ecteinascidins and related compounds

ABSTRACT

Ecteinascidin compounds with a quinone ring for ring E are active as anti-cancer agents. Related processes and compounds are provided.

FIELD OF THE INVENTION

The present invention relates to synthetic processes, compounds obtainedwith these processes and their use as antitumor agents. In particular itrelates to synthetic processes for producing naturally occurringecteinascidin compounds and related analogues, including novelintermediates that form a part of such synthetic processes.

In addition, the present invention relates to novel, previouslyundisclosed indications of the ecteinascidin analogues.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,089,273, describes novel compositions of matterextracted from the tropical marine invertebrate, Ecteinascidiaturbinata, and designated therein as ecteinascidins 729, 743, 745, 759A,759B and 770. These compounds are useful as antibacterial and/orantitumor agents in mammals. Ecteinascidin 743 is undergoing clinicaltrials as an antitumour agent.

The limited availability of natural material has resulted in the searchfor alternative synthetic methods being sought for the natural compoundsand related analogs.

A synthetic process for producing ecteinascidin compounds is describedin U.S. Pat. No. 5,721,362. The claimed method involves many steps,there being 38 Examples each describing one or more steps in thesynthetic sequence to arrive at ecteinascidin 743.

Shorter synthetic processes for producing Ecteinascidin 743 aredescribed in WO 0069862 and WO 0187895 and involve the use ofcyanosafracin B as starting material.

However, there is still a need to provide synthetic routes to otherecteinascidins, in particular to provide more economic paths to theknown antitumour agents such as ET-729, as well as permitting thepreparation of new compounds.

Synthetic ecteinascidin compounds are known from various earlier PCTfilings, including for example WO 0018233, WO 0177115, WO 0187894, WO0187895, WO 99 51238, and WO 9846080. All of these patent specificationsare specifically incorporated by reference, especially for the guidancethey give in the design and synthesis of ecteinascidin compounds. Inparticular, they reveal structure-activity relationships which may beapplied to the compounds of the present invention. See also J. Am. Chem.Soc, 1996, vol. 118, no. 38, pages 9017-9023 for ecteinascidincompounds. The synthetic compounds and the natural ecteinascidins have afused ring system:

In many ecteinascidins, there is a 1,4 bridge across the fused ringsystem. With the natural ecteinascidins, the 1,4 bridge is sometimes a1,4-spiroamine bridge, as for instance in ecteinascidin 729, 736 or 743.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides quinone ecteinascidincompounds having a quinone group in ring E. Such compounds are typicallyof the formula (Ab):

where R^(a), R^(b), R⁵, R⁷, R⁸, R²¹, X and ring E are as defined, thesulphur in the 1,4 bridge may be oxidised, and R^(12b) is as defined forR¹² or R^(12a).

These quinone compounds are of special interest for their activity,notably the activity of ecteinascidin 637 quinone. They can be made byoxidation of an ecteinascidin compound having a ring E which isphenolic, as for example in ecteinascidin 743 with a 16-methyl,17-methoxy, 18-hydroxy phenyl ring for ring E. Other substituents can beemployed. Suitable oxidising agents include Fremy's salt.

In a related aspect, the present invention provides a process for makingecteinascidin 729 and related compounds having a hydrogen at the N-12position. To this end, the present invention provides a process whichcomprises providing an ecteinascidin with a substituent at the N-12position, and removing that substituent. Thereafter the N—H group at the12-position can be derivatised, for example with a group R^(12a).

The invention also provides a process which is a modification of theprocess for preparing an ecteinascidin product as described in our WO0187895. Thus, the present invention provides a process for preparing anecteinascidin compound wherein a 1,4 bridge is formed using a 1-labile,10-hydroxy, 12-protected, 18-protected hydroxy, di-6,8-enone fused ringprecursor compound.

The 1,4 bridge can be a spiroamine, for example as in ecteinascidin 729,but need not be such a group. Typically the 1,4 bridge is of theformula:—⁽¹⁾CH₂—X—C(═O)—C(R^(a))(R^(b))—S⁽⁴⁾—where the —CH₂— is at the 1-position and the —S— is at the 4-position,of the ecteinascidin compound, with the groups X, R^(a) and R^(b) beingas herein defined.

As a related part of this invention, the invention provides anecteinascidin compound which is of the formula (A):

wherein:R^(a) and R^(b) together with the carbon to which they are attached forma group C(═O)—; a group —CH(R^(c))— where R^(c) is OX¹ or N(X¹X²) wherethe or each X¹, X² is independently H, —C(═O)R′, substituted orunsubstituted hydrocarbyl; or a spiro ring;R⁵ is —OH or a protected or derivatised version of such a group;R⁷ is —OCH₃ and R⁸ is —OH or R⁷ and R⁸ together form a group —O—CH₂—O—;R¹² is a protecting group;R²¹ is —H, —OH or —CN;X is —NH— or —O—;ring E is of the formula:

where R¹⁸ is —OH or a protected or derivatised version of such a group;and the sulphur in the 1,4 bridge may be oxidised.

The 1,4-bridge can be omitted, especially for the new quinone compounds.In that instance, the subsituent at the 1-position, R¹, can be as in ourWO 0187894.

The protecting group on N-12 can be removed to give a hydrogen, andoptionally replaced with another substituent to give other compounds ofthis invention. Examples of such derivatised compounds include thosewhere the group at N-12 is alkyl such as methyl or ethyl, especiallymethyl, or is acyl, especially acetyl.

In this respect, the present invention further provides compounds offormula (Aa):

where R^(a), R^(b), R⁵, R⁷, R⁸, R²¹, X and ring E are as defined, thesulphur in the 1,4 bridge may be oxidised, and R^(12a) is hydrogen,substituted or unsubstituted hydrocarbyl, or substituted orunsubstituted acyl, but is preferably not methyl.

In another aspect, the present invention provides ecteinascidincompounds having a 1,4 bridge where the oxygen β to the 1-position isreplaced by an isostere. Suitable isosteres include —NH—.

Thus, according to the present invention, there are provided 1,4-bridgedecteinascidin compounds wherein the 1,4 bridge is of the formula:—⁽¹⁾CH₂—X—C(═O)—C(R^(a))(R^(b))—S⁽⁴⁾—where the —CH₂— is at the 1-position, and the —S— is at the 4-position,of the ecteinascidin compound, with the group X being NH, and R^(a) andR^(b) being as herein defined.

Such compounds include those of the formula (Ac):

where R^(a), R^(b), R⁵, R⁷, R⁸, R^(12b), R²¹, and ring E are as definedand the sulphur in the 1,4 bridge may be oxidised.

These compounds can be prepared by a process of this invention which isa modification of the process of WO 0187895, with a 1-labile substituentwhich is suitably a group of formula:—CH₂—NProt¹-C(═O)—CHNProt²—S—Hwhere Prot¹ and Prot² are amine protecting groups.

The protecting groups can then be removed separately or together, andthe respective nitrogen atoms derivatised as appropriate.

Suitable procedures can be devised in the light of the disclosure in WO0187894, WO 0187895, WO 0177115, which are incorporated herein byspecific reference.

PREFERRED EMBODIMENTS

The quinone compounds of this invention are preferably made by a processwhich involves oxidation of an ecteinascidin having a phenol for ring E,where the hydroxy function of the phenol is at position 18 and may besubstituted.

Such a reaction be in accordance with the following scheme:

Alternatively, the quinone compounds of this invention can be made bymodification of the synthetic procedures known from our earlier patentapplications and which start from safracin B or a related compound. Inparticular, the present invention provides a process based on thatdisclosed in our WO 0069862 where ring E is a quinone ring in thestarting material, and the ring E is not converted into the phenolsystem, as mentioned at the bottom of printed page 24 of the WO00699862.

The 1,4 bridge need not be present in the quinone compounds.

In the compounds of this invention with a 1,4-bridge, preferred examplesof the group formed by R^(a) and R^(b) together with the carbon to whichthey are attached include:

—C(═O)—;

—CHNH₂ or a protected or derivatised version of such a group;

—CHOH or a protected or derivatised version of such a group;

a group of formula:

where R^(d) and X¹ are as defined;

a group of formula:

where R^(d) and X¹ are as defined.

R^(d) and X¹ in these groups are preferably chosen from hydrogen orsubstituted or unsubstituted R′, OR′, —(C═O)R′, hydrocarbyl,hydrocarbyloxy or hydrocarboyl, especially hydrogen, unsubstituted orsubstituted alkyl or alkoxy, unsubstituted or substituted alkenyl,substituted or unsubstituted alkynyl, unsubstituted or substituted aryl,unsubstituted or substituted aralkyl; preferably hydrogen, alkyl oralkoxy, more preferably hydrogen, methyl or methoxy, most preferablyboth hydrogen.

Preferred definitions include those which give a group of formula:

where R^(d) and X¹ are as defined; ora group of formula:

where R^(d) and X¹ are as defined.

In particular, R^(a) and R^(b) can be chosen to give a group of formula:

Where R^(a) and R^(b) together with the carbon to which they areattached form a group —CHNH₂ or a protected or derivatised version ofsuch a group, the group can be of the formula —CHNHX₁ or —CHN(X₁X₂)where X₁, X₂ is H, C(═O)R′, substituted or unsubstituted C₁-C₁₈ alkyl,substituted or unsubstituted C₂-C₁₈ alkenyl, substituted orunsubstituted C₂-C₁₈ alkynyl, substituted or unsubstituted aryl, or aprotecting group. Preferred definitions include hydrogen, acyl such asacetyl or a protecting group.

One of R^(a) or R^(b) is often hydrogen and the other is preferably H;—NHCOalkyl, particularly where the alkyl has up to 16 carbon atoms, suchas 1, 4, 7, 15 carbon atoms and may be halo substituted optionallyperhalosubstituted; —NHalkylCOOH particularly where the alkyl has up to4 carbon atoms; protected —NHCOCH(NH₂)CH₂SH where the NH₂ and/or the SHare protected; —NHbiotin; —NHaryl; —NH(aa)y where aa is an amino acidacyl and y is suitably 1, 2 or 3 and wherein any NH₂ is optionallyderivatised or protected, as with an amide terminal group or a Docgroup; phthalimido formed —NX₂—; alkyl preferably having 1 to 4 carbonatoms; arylalkenyl, especially cinnamoyl which may be substituted aswith 3-trifluoromethyl;

Preferred examples of the group R^(a) or R^(b) include NHAc,NHCO(CH₂)₂COOH, NHCOCH(NHAlloc)CH₂SFm, NHCO(CH₂)₁₄CH₃, NHTFA,NHCO(CH₂)₂CH₃, NHCOCH₂CH(CH₃)₂, NHCO(CH₂)₆CH₃, NHBiotin, NHBz, NHCOCinn,NHCO-(p-F₃C)-Cinn, NHCOVal-NH₂, NHCOVal-N—Ac, NHCOVal-N—COCinn,NHCOVal-Ala-NH₂, NHCOVal-Ala-N—Ac, NHCOAla-NH₂, OH, OAc, NHAc,NHCO(CH₂)₂COOH, NHCOCH(NHAlloc)CH₂SFm, NHCOCH(NH₂)CH₂SFm, NPhth,NH-(m-CO₂Me)-Dz, NHCO(CH₂)₁₄CH₃, NMe₂, NHTFA, NHCO(CH₂)₂CH₃,NHCOCH₂CH(CH₃)₂, NHCO(CH₂)₆CH₃, NHAlloc, NHTroc, NHBiotin, NHBz,NHCOCinn, NHCO—(P—F₃C)-Cinn, NHCOVal-NH₂, NHCOVal-N—Ac,NHCOVal-N—COCinn, NHCOVal-Ala-NH₂, NHCOVal-Ala-N—Ac,NHCOVal-Ala-N—COCinn, NHCOAla-NH₂, NHCOAla-N—Ac, NHCOAla-N—COCinn, OH,OAc, NHAc, NHCO(CH₂)₂COOH, NHCOCH(NHAlloc)CH₂SFm, Nphth, along withsimilar groups where the number of carbon atoms is varied or the aminoacid is changed or another change of this kind is made to give a similargroup.

Where R^(a) and R^(b) together with the carbon to which they areattached form a group —CHOH or a protected or derivatised version ofsuch a group, the group can be of the formula —CHOX₁, where X₁ is asdefined.

Other preferred examples include OH, OAc, OCOCF₃, OCOCH₂CH₂CH₃,OCO(CH₂)₆CH₃, OCO(CH₂)₁₄CH₃, OCOCH═CHPh, —OSO₂CH₃ along with similargroups where the number of carbon atoms is varied or differentsubstituent groups are introduced or another change of this kind is madeto give a similar group.

The sulphur in the 1,4 bridge may be oxidised to give, for example agroup —S(═O)—.

Where the 1,4 bridge is not present, the group at the 1-position, R¹, issuitably an optionally protected or derivatised aminomethylene group,oran optionally protected or derivatised hydroxymethylene group; and thegroup at the 4-position, R⁴, is typically hydrogen.

R¹ is suitably a hydrophobic group and which thus lacks free amino,hydroxy or other hydrophilic function. Typically R¹ is a group—CH₂—NH₂—CO—R′, where R′ is as defined but preferably has a linear chainlength of less than 20 atoms, more preferably less than 15 or 10 atoms,where a 1,4-phenyl is counted as a chain length of four atoms andsimilar considerations apply to other cyclic groups (for example,1,2-cyclohexyl is chain length of two), and the linear chain of lessthan 10, 15 or 20 atoms can itself be substituted. In particular, datasuggests there is a balance to be achieved between having no such groupRa—CO— and having a large, bulky group.

In particularly preferred compounds, the group R¹ is acylated on an —NH₂group, and for example N-acyl derivatives can be formed from groups—CH₂NH₂ and —CH₂—NH-aa, where aa is amino acid. The acyl derivatives canbe N-acyl or N-thioacyl derivatives thereof. The acyl groups can be offormula —CO—R′, where R′ is as defined and is chosen to meet theindicated criteria. Suitable acyl groups include alanyl, arginyl,aspartyl, asparagyl, cystyl, glutamyl, glutaminyl, glycyl, histidyl,hydroxyprolyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl,prolyl, seryl, threonyl, thyronyl, tryptophyl, tyrosyl, valyl, as wellas other amino acid acyl groups, which may be L- or D-. Such amino acidacyl groups are preferred derivatised on the amino group to givehydrophobicity.

In a variation, the group R¹ is a derivatised hydroxymethylene group.Similar considerations apply as with the derivatised aminomethylenegroup.

In one preferred aspect, at least one of R⁵, R¹⁸ and R′ in the groupR^(d) is selected from hydrogen, R′, C═OR′, or COOR′, where R′ isoptionally substituted alkyl or alkenyl, the optional substituents beingchosen from halo, amino including amino derived from amino acid, aryl orheterocyclic.

R⁵ is preferably —OH or a protected or derivatised version of such agroup. In particular, it can be a group —OX₁. Particularly preferred forR⁵ is an acyloxy group, especially an acetyloxy group. Other examplesinclude cinnamoyloxy and heptanoyloxy.

R⁷ is —OCH₃ and R⁸ is —OH or more preferably R⁷ and R⁸ together form agroup —O—CH₂—O—.

R¹² is a protecting group for the nitrogen atom of the amine function.Suitable protecting groups for such amines include carbamates, amides,and other protecting groups, such as alkyl, arylalkyl, sulpho- orhalo-arylalkyl, haloalkyl, alkylsilylalkyl, arylalkyl, cycloalkylalkyl,alkylarylalkyl, heterocyclylalkyl, nitroarylalkyl, acylaminoalkyl,nitroaryldithioarylalkyl, dicycloalkylcarboxamidoalkyl, cycloalkyl,alkenyl, arylalkenyl, nitroarylalkenyl, heterocyclylalkenyl,heterocyclyl, hydroxyheterocyclyl, alkyldithio, alkoxy- or halo- oralkylsulphinyl arylalkyl, hetercyclylacyl, and other carbamates, andalkanoyl, haloalkanoyl, arylalkanoyl, alkenoyl, heterocyclylacyl, aroyl,arylaroyl, haloaroyl, nitroaroyl, and other amides, as well as alkyl,alkenyl, alkylsilylalkoxyalkyl, alkoxyalkyl, cyanoalkyl, heterocyclyl,alkoxyarylalkyl, cycloalkyl, nitroaryl, arylalkyl, alkoxy- orhydroxy-arylalkyl, and many other groups. Such groups may optionally besubstituted. Further examples are given in our earlier patentspecifications.

A preferred class of compounds comprise an ecteinascidin compoundwherein the N-12 protecting group R¹² is chosen from haloalkyl,alkoxyalkyl, haloalkoxyalkyl, arylalkylene, haloalkylarylakylene, acyl,haloacyl, optionally halo alkoxyalkyl, optionally halo or alkylarylalkenylacyl, alkenylacyl, carbonate, carbamate, arylalkyl, alkenyl,acid anhydride and amino acid.

Especially preferred is an ecteinascidin compound wherein the N-12protecting group R¹² is chosen from allyl, acetyl, trifluoroacetyl,2,2,2-trichloroethoxycarbonyl, isovalerylcarbonyl,trans-3-(trifluoromethyl)cinnamoylcarbonyl, heptafluorobutrylcarbonyl,decanoylcarbonyl, trans-cinnamoylcarbonyl, butyrylcarbonyl,3-chloropropionylcarbonyl, cinnamoylcarbonyl, 4-methylcinnamoylcarbonyl,hydrocinnamoylcarbonyl, or trans-hexenoylcarbonyl, or alanyl, arginyl,aspartyl, asparagyl, cystyl, glutamyl, glutaminyl, glycyl, histidyl,hydroxyprolyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl,prolyl, seryl, threonyl, thyronyl, tryptophyl, tyrosyl, valyl, or otheramino acid acyl group, phthalimido or other cyclic amido group.

R¹⁸ is suitably as defined for R⁵, though the most preferred definitionis hydroxy.

R²¹ is —H, or more preferably —OH or CN.

Ring E is of the formula:

where R¹⁸ is —OH or a protected or derivatised version of such a groupand is suitably of the formula —OX₁. Examples apart from —OH includecinnamoyloxy.

X₁ or X₂ when an amine protecting group, and Prot¹ and Prot² can be asdefined for R¹², and reference is made to WO 0187895 for moreinformation.

X₁ when a hydroxy protecting group, and Prot³ can be known protectinggroups for a hydroxy function. Suitable protecting groups for hydroxygroups include ethers and esters, such as alkyl, alkoxyalkyl,aryloxyalkyl, alkoxyalkoxyalkyl, alkylsilylalkoxyalkyl, alkylthioalkyl,arylthioalkyl, azidoalkyl, cyanoalkyl, chloroalkyl, heterocyclic,arylacyl, haloarylacyl, cycloalkylalkyl, alkenyl, cycloalkyl,alyklarylalkyl, alkoxyarylalkyl, nitroarylalkyl, haloarylalkyl,alkylaminocarbonylarylalkyl, alkylsulfinylarylalkyl, alkylsilyl andother ethers, and arylacyl, aryl alkyl carbonate, aliphatic carbonate,alkylsulfinylarlyalkyl carbonate, alkyl carbonate, aryl haloalkylcarbonate, aryl alkenyl carbonate, aryl carbamate, alkyl phosphinyl,alkylphosphinothioyl, aryl phosphinothioyl, aryl alkyl sulphonate andother esters. Such groups may optionally be substituted ether examplesare given in our earlier patent specifications.

Each group R′ is independently selected from the group consisting of H,OH, NO₂, NH₂, SH, CN, halogen, ═O, C(═O)H, C(═O)CH₃, CO₂H, CO₂CH₃, C₁-C₆alkyl, alkynyl, alkenyl, aryl, aralkyl and heterocyclic.

Preferred definitions include H, acyl, alkyl, especially H and alkanoylor cinnamoyl.

Preferred compounds of this invention include those complying with oneor more of the following definitions:R¹ is —CH₂NH₂ or —CH₂OH, or a protected or derivatised version of such agroup (for which especially see WO 0187894, incorporated by specificreference at this point for its teaching of R¹ and thus all the teachingin WO 0187894 on R¹ forms part of the present text) and R⁴ is —H; or R¹and R⁴ together form a group of formula (II), (III), (IV), (V) or (VI)wherein X is O, NH or NR and Y is O, S or S═O and R is a nitrogenprotecting group and R′ is H or OH or OMe or Me.

R⁵ is —OH, —OAc or —OAllyl or —OCinnamoyl or —OOctanoyl;R⁷ and R⁸ together form a group —O—CH₂—O—;R^(12b) is H, alkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl,arylalkylene, haloalkylarylakylene, acyl, haloacyl, carbonate,carbamate, arylalkyl, alkenyl and amino acid. Preferably R^(12b) is H,methyl, allyl, acetyl, trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl,isovalerylcarbonyl, trans-3-(trifluoromethyl)cinnamoylcarbonyl,heptafluorobutrylcarbonyl, decanoylcarbonyl, trans-cinnamoylcarbonyl,butyrylcarbonyl, 3-chloropropyonylcarbonyl, cinnamoylcarbonyl,4-methylcinnamoylcarbonyl, hydrocinnamoylcarbonyl, ortrans-hexenoylcarbonyl, or alanyl, arginyl, aspartyl, asparagyl, cystyl,glutamyl, glutaminyl, glycyl, histidyl, hydroxyprolyl., isoleucyl,leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, threonyl,thyronyl, tryptophyl, tyrosyl, valyl, as well as other less common aminoacid acyl groups, as well as phthalimido and other cyclic amides.Ring E is of the formula:

Suitable halogen substituents in the compounds of the present inventioninclude F, Cl, Br and I.

Alkyl groups preferably have from 1 to 24 carbon atoms. One morepreferred class of alkyl groups has 1 to about 12 carbon atoms, yet morepreferably 1 to about 8 carbon atoms, still more preferably 1 to about 6carbon atoms, and most preferably 1, 2, 3 or 4 carbon atoms. Anothermore preferred class of alkyl groups has 12 to about 24 carbon atoms,yet more preferably 12 to about 18 carbon atoms, and most preferably 13,15 or 17 carbon atoms. Methyl, ethyl and propyl including isopropyl areparticularly preferred alkyl groups in the compounds of the presentinvention. As used herein, the term alkyl, unless otherwise modified,refers to both cyclic and noncyclic groups, although cyclic groups willcomprise at least three carbon ring members.

Preferred alkenyl and alkynyl groups in the compounds of the presentinvention have one or more unsaturated linkages and from 2 to about 12carbon atoms, more preferably 2 to about 8 carbon atoms, still morepreferably 2 to about 6 carbon atoms, even more preferably 1, 2, 3 or 4carbon atoms. The terms alkenyl and alkynyl as used herein refer to bothcyclic and noncyclic groups, although straight or branched noncyclicgroups are generally more preferred.

Preferred alkoxy groups in the compounds of the present inventioninclude groups having one or more oxygen linkages and from 1 to about 12carbon atoms, more preferably from 1 to about 8 carbon atoms, and stillmore preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3or 4 carbon atoms.

Preferred alkylthio groups in the compounds of the present inventionhave one or more thioether linkages and from 1 to about 12 carbon atoms,more preferably from 1 to about 8 carbon atoms, and still morepreferably 1 to about 6 carbon atoms. Alkylthio groups having 1, 2, 3 or4 carbon atoms are particularly preferred.

Preferred alkylsulfinyl groups in the compounds of the present inventioninclude those groups having one or more sulfoxide (SO) groups and from 1to about 12 carbon atoms, more preferably from 1 to about 8 carbonatoms, and still more preferably 1 to about 6 carbon atoms.Alkylsulfinyl groups having 1, 2, 3 or 4 carbon atoms are particularlypreferred.

Preferred alkylsulfonyl groups in the compounds of the present inventioninclude those groups having one or more sulfonyl (SO²) groups and from 1to about 12 carbon atoms, more preferably from 1 to about 8 carbonatoms, and still more preferably 1 to about 6 carbon atoms.Alkylsulfonyl groups having 1, 2, 3 or 4 carbon atoms are particularlypreferred.

Preferred aminoalkyl groups include those groups having one or moreprimary, secondary and/or tertiary amine groups, and from 1 to about 12carbon atoms, more preferably 1 to about 8 carbon atoms, still morepreferably 1 to about 6 carbon atoms, even more preferably 1, 2, 3 or 4carbon atoms. Secondary and tertiary amine groups are generally morepreferred than primary amine moieties.

Heterocylic groups include heteroaromatic and heteroalicyclic groups.Suitable heteroaromatic groups in the compounds of the present inventioncontain one, two or three heteroatoms selected from N, O or S atoms andinclude, e.g., coumarinyl including 8-coumarinyl, quinolinyl including8-quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl,thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl and benzothiazol.Suitable heteroalicyclic groups in the compounds of the presentinvention contain one, two or three heteroatoms selected from N, O or Satoms and include, e.g., tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolindinyl groups.

Suitable carbocyclic aryl groups in the compounds of the presentinvention include single and multiple ring compounds, including multiplering compounds that contain separate and/or fused aryl groups. Typicalcarbocyclic aryl groups contain 1 to 3 separate or fused rings and from6 to about 18 carbon ring atoms. Specifically preferred carbocyclicarykl groups include phenyl including substituted phenyl, such as2-substituted phenyl, 3-substituted phenyl, 2,3-substituted phenyl,2,5-substituted phenyl, 2,3,5-substituted and 2,4,5-substituted phenyl,including where one or more of the phenyl substituents is anelectron-withdrawing group such as halogen, cyano, nitro, alkanoyl,sulfinyl, sulfonyl and the like; naphthyl including 1-naphthyl and2-naphthyl; biphenyl; phenanthryl; and anthracyl.

Preferred acyl groups are R′—CO— including alkyl-CO, alkenyl-CO,alkynyl-CO, aryl-CO, heterocyclic-CO, amongst others.

References herein to substituted groups in the compounds of the presentinvention refer to the specified moiety that may be substituted at oneor more available positions by one or more suitable groups as mentionedabove, e.g., halogen such as fluoro, chloro, bromo and iodide; cyano;hydroxyl; nitro; azido; alkanoyl such as a C1-6 alkanoyl group such asacyl and the like; carboxamido; alkyl groups including those groupshaving 1 to about 2 carbon atoms or from 1 to about 6 carbon atoms andmore preferably 1-3 carbon atoms; alkenyl and alkynyl groups includinggroups having one or more unsaturated linkages and from 2 to about 12carbon or from 2 to about 6 carbon atoms; alkoxy groups having thosehaving one or more oxygen linkages and from 1 to about 12 carbon atomsor 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groupsincluding those moieties having one or more thioether linkages and from1 to about 12 carbon atoms or from 1 to about 6 carbo atoms;alkylsulfinyl groups including those moieties having one or moresulfinyl linkages and from 1 to about 12 carbon atoms or from 1 to about6 carbon atoms; alkylsulfinyl groups including those moieties having oneor more sulfonyl linkages and from 1 to about 12 carbon atoms or from 1to about 6 carbon atoms; aminoalkyl groups such as groups having one ormore N atoms and from 1 to about 12 carbon atoms or from 1 to about 6carbon atoms; carbocyclic aryl having 6 or more carbons, particularlyphenyl (e.g., R being a substituted or unsubstituted biphenyl moiety);and aralkyl such as benzyl.

Preferred R′ groups are present in groups of formula R′, COR′ or OCOR′and include alkyl or alkenyl, that may be substituted at one or moreavailable positions by one or more suitable groups, e.g., halogen suchas fluoro, chloro, bromo and iodo, especially ω-chloro or perfluoro;aminoalkyl groups such as groups having one or more N atoms and from 1to about 12 carbon atoms or from 1 to about 6 carbon atoms, andespecially including amino acid, notably glycine, alanine, arginine,asparagine, asparaginic acid, cystein, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine or valine, especiallyprotected forms of such amino acids; carbocylic aryl having 6 or morecarbons, particularly phenyl; and aralkyl such as benzyl; heterocyclicgroups including heteroalicyclic and heteroaromatic groups, especiallywith 5 to 10 ring atoms of which 1 to 4 are heteroatoms, more preferablyheterocyclic groups with 5 or 6 ring atoms and 1 or 2 heteroatoms orwith 10 ring atoms and 1 to 3 heteroatoms, the heterocyclic groupsoptionally being substituted with one or more of the substituentspermitted for R′ and especially amino such as dimethylamino or withketo.

The acyl derivatives such as CO—R′ can be N-acyl or N-thioacylderivatives thereof, as well as cyclic amides. The acyl groups canillustratively be alkanoyl, haloalkanoyl, arylalkanoyl, alkenyl,heterocyclylacyl, aroyl, arylaroyl, haloaroyl, nitroaroyl, or other acylgroups. R′ or similar group of an acyl can be various groups such asalkyl, alkoxy, alkylene, arylalkyl, arylalkylene, amino acid acyl, orheterocyclyl, each optionally substituted with halo, cyano, nitro,carboxyalkyl, alkoxy, aryl, aryloxy, heterocyclyl, heterocycyloxy,alkyl, amino or substituted amino. Other acylating agents includeisothiocyanates, such as aryl isothiocyanates, notably phenylisocyanate. The alkyl, alkoxy or alkylene groups suitably have 1 to 6 or12 carbon atoms, and can be linear, branched or cyclic. Aryl groups aretypically phenyl, biphenyl or naphthyl. Hetercyclyl groups can bearomatic or partially or completely unsaturated and suitably have 4 to 8ring atoms, more preferably 5 or 6 ring atoms, with one or moreheteroatoms selected from nitrogen, sulphur and oxygen.

Without being exhaustive, typical R′ groups in acyl groups includealkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, arylalkylene,haloalkylarylakylene, acyl, haloacyl, arlyalkyl, alkenyl and amino acid.For example, R′—CO— can be acetyl, trifluoroacetyl,2,2,2-trichloroethoxycarbonyl, isovalerylcarbonyl,trans-3-(trifluoromethyl)cinnamoylcarbonyl, heptafluorobutyrylcarbonyl,decanoylcarbonyl, trans-cinnamoylcarbonyl, butyrylcarbonyl,3-chloropropyonylcarbonyl, cinnamoylcarbonyl, 4-methylcinnamoylcarbonyl,hydrocinnamoylcarbonyl, or trans-hexenoylcarbonyl, or alanyl, arginyl,aspartyl, asparagyl, cystyl, glutarnyl, glutaminyl, glycyl, histidyl,hydroxyprolyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl,prolyl, seryl, threonyl, thyronyl, tryptophyl, tyrosyl, valyl, as wellas other less common amino acid acyl groups, as well as phthalimido andother cyclic amides.

One class of preferred compounds of this invention includes compounds ofthis invention which have one or more of the following substituents:

R¹ and R⁴ form a bridge as defined, or R¹ is as defined and R⁴ ishydrogen.

R⁵ is hydrogen;

alkyl, more preferably alkyl of 1 to 6 carbon atoms;

C(═O)R′, where R′ is alkyl, more preferably alkyl of 1 to 24 carbonatoms, especially 1 to 8 or 12 to 18 carbon atoms; haloalkyl, morepreferably ω-chloro- or perfluoro-alkyl of 1 to 4 carbon atoms,especially ω-chloroethyl or perfluoromethyl, ethyl or propyl;heterocylicalkyl, more preferably an aylkyl of 1 to 6 carbon atoms withan ω-heterocyclic substituent suitably having 5 to 10 ring atoms and 1to 4 heteroatoms, including fused heteroalicyclic with 3 hetero atoms,such as biotin; aminoalkyl, more preferably alkyl of 1 to 6 carbonatoms, especially 2 carbon atoms, with an ω-amino group optionallyprotected for example with alkoxycarbonyl such as (CH₃)₃C—O—C═O— orother protecting group;

arylalkylene, especially cinnamoyl; alkylene, especially vinyl or allyl;aralkyl, such as benzyl; or

C(═O)OR′, where R′ is alkyl, more preferably alkyl of 1 to 6 carbonatoms, especially branched alkyl; alkenyl, more preferably allyl;

R¹² is hydrogen, methyl, or a protecting group including alkoxycarbonylsuch as (CH₃)₃C—O—C═O—.

R¹⁸ when present is hydrogen;

alkyl, more preferably alkyl of 1 to 6 carbon atoms;

(C═O)R′, where R′ is alkoxy, especially with an alkyl group of 1 to 6carbon atoms; alkyl, more preferably alkyl of 1 to 24 carbon atoms,preferably 1 to 8 or 12 to 18 carbon atoms; haloalkyl, more preferablyperfluoroalkyl of 1 to 4 carbon atoms, especially perfluoromethyl, ethylor propyl; arylalkylene, especially cinnamoyl; heterocylicalkyl, morepreferably an alkyl of 1 to 6 carbon atoms with an ω heterocyclicsubstituent suitably having 5 to 12 ring atoms and 1 to 4 heteroatoms,including fused heterocyclic with 3 ring atoms, such as biotin;heterocyclicalkyl, with preferably 1 carbon atom in the alkyl group, andmore preferably heteroalicylicmethyl with 5 to 10 ring atoms and 1 to 4ring atoms, especially fused heterocylic with 1 to 4 heteroatoms, suchas dimethylaminocoumarin or coumarin; alkylene, especially allyl;aralkyl, such as benzyl;

(C═O)OR′, where R′ is alkyl, more preferably alkyl of 1 to 6 carbonatoms; alkylene, especially vinyl or allyl; aralkyl, such as benzyl.

R^(d) is OC(═O)R′, where R′ is allyl, more preferably alkyl of 1 to 24carbon atoms, preferably 1 to 8 or 12 to 18 carbon atoms; haloalkyl,more preferably ω-chloro- or perfluoro-alkyl of 1 to 4 carbon atoms,especially ω-chloroethyl or perfluoromethyl, ethyl or propyl; aralkyl,such as benzyl or phenethyl; arylalkylene, especially cinnamoyl;aminoalkyl, especially amino acid, more especially protected amino acid,including protected cysteinine, notably Fm—S CH₂CH(NHBOC)-cys orprotected alanine, notably (CH₃)₃C—O—C═O-ala; heterocyclicalkyl, morepreferably an alkyl of 1 to 6 carbon atoms with an ω-heterocyclicsubstituent suitably having 5 to 12 ring atoms and 1 to 4 heteroatoms,including fused heterocyclic with 3 ring atoms, such as biotin;heterocyclicalkyl, with preferably 1 carbon atom in the alkyl group, andmore preferably heteroalicyclicmethyl with 5 to 10 ring atoms and 1 to 4ring atoms, especially fused heterocylic with 1 to 4 heteroatoms, suchas coumarin or dimethylaminocoumarin;

O(C═O)OR′, where R′ is alkyl, more preferably alkyl of 1 to 6 carbonatoms; alkylene, especially vinyl or allyl; aralkyl, such as benzyl;

OP═O(OR′)₂, where R′ is benzyl.

X¹ is hydrogen;

alkyl, more preferably alkyl of 1 to 6 carbon atoms;

(C═O)OR′, where R′ is alkylene, especially vinyl.

R²¹ is hydrogen, hydroxy, or cyano.

Compounds with changes at R⁵ are part of this invention, especiallyester groups, R₁=R′CO—, with R′ a long aliphatic or aromatic group.

There are compounds that have good ADME properties(absorption-distribution-metabolism-excretion) which are good indicativeof pharmacokinetics.

In a related aspect of this invention, the compounds have one or more ofthe following features:

R¹ is —CH₂—N(R′)₂ or —CH₂—OR′, where each R′ is H; alkyl-CO—;haloalkyl-CO—; cycloalkylalkyl-CO—; haloalkyl-O—CO—; arylalkyl-CO—;arylalkenyl-CO—; heteroaryl-CO—; alkenyl-CO—; alkenyl; amino acid acyl;or a protecting group;

R⁵ is acetyl or another acyl. Preferably it has at least 4, 5 or 6carbon atoms, for example up to 18 or 24 carbon atoms. Suitablesubstituents include esters COR′, where R′ is alkyl, alkenyl, often withone or more substituents. Alkyl, substituted alkyl, alkenyl andarylalkenyl are preferred, with suitable substituents including aryl,heterocyclic. Other definitions for R⁵ include esters of formula COR′derived from an amino acid, optionally a protected amino acid.

R¹⁸ is hydroxy or it is OR′, OCOR′ or OCOOR′ where R′ is a substituentwith some bulk. Such bulky substituents include those with branchedchain groups, unsaturated groups or cyclic groups including aromaticgroups. Thus, branched alkyl, cycloalkyl, branched alkenyl, aryl,heteroaromatic and related groups are preferred for inclusion within thestructure of the substituent R¹⁸. Preferably the total number of carbonatoms is 2 to 24, more preferably 6 to 18 carbon atoms. Typically R¹⁸ isan ester, ether or carbonate, being of formula OCOR′, OR′ or OCOOR′.

R^(d) is hydroxy or methoxy. Alternatively, it is OR′, OCOR′ or OCOOR′where R′ is a substituent with some bulk. Such bulky substituentsinclude those with branched chain groups, unsaturated groups or cyclicgroups including aromatic groups. Thus, branched alkyl, cycloalkyl,branched alkenyl, aryl, heteroaromatic and related groups are preferredfor inclusion within the structure of the substituent. Preferably thetotal number of carbon atoms is 2 to 24, more preferably 6 to 18 carbonatoms.

Without being exhaustive, another class of preferred compounds of thisinvention have one or more of the following definitions: R²¹ is H, —CNor —OH, most especially —OH or —CN.

R⁵ is preferably H or acetyl; arylalkyl, especially benzyl;alkyl-CO-(alkyl being up to 25 carbon atoms, such as up to 17, 19 or 21carbon atoms and preferably an odd number of carbon atoms correspondingto a fatty acid carboxylic acid of even number of carbon atoms or else alow number of carbon atoms such as 1 to 6) especially CH₃—(CH₂)_(n)—CO—where n is for example 1.2.4.6.12.14 or 16; haloalkyl-CO—, especiallytrifluoromethylcarbonyl; arylalkyl-CO—, especially benzyl-CO—;arylalkenyl-CO—, especially cinnamoyl-CO—; most especially R₁ is H,acetyl or cinnamoyl.

R¹² is H; alkyl, especially methyl; alkyl-O—CO—, especiallyt-butyl-O—CO— or alkenyl-O—CO—, especially allyl-O—CO—.

R¹⁸ is preferably H or acetyl; alkyl (alkyl being 1 to 6 carbon atoms),especially C₁ to C₃ alkyl; alkenyl, especially allyl; arylalkyl,especially benzyl; alkyl-CO— (alkyl being up to 25 carbon atoms, such asup to 17, 19 or 21 carbon atoms and preferably an odd number of carbonatoms corresponding to a fatty acid carboxylic acid of even number ofcarbon atoms or else a low number of carbon atoms such as 1 to 6)especially CH₃—(CH₂)_(n)—CO— where n is for example 1.2.4.6.12.14 or 16and derivatives thereof, as in Biotin-(CH₂)₄—CO—; arylalkenyl-CO—,especially cinnamoyl-CO—; alkyl-O—CO—, especially t-butyl-O—CO—;arylalkyl-O—CO—, especially benzyl-O—CO—; alkenyl-O—CO, especiallyallyl-O—CO—.

R^(d) is preferably OH, O-acetyl, O-allyl (alkyl being 1 to 6 carbonatoms) especially C₁ to C₃ alkyl; O-alkenyl, especially allyl;arylalkyl-O—, especially benzyl; alkyl-CO—O— (alkyl being up to 25carbon atoms, such as up to 17, 19 or 21 carbon atoms and preferably anodd number of carbon atoms corresponding to a fatty acid carboxylic acidof even number of carbon atoms or else a low number of carbon atoms suchas 1 to 6) especially CH₃—(CH₂)_(n)—CO—O— where n is for example 1, 2,4, 6, 12, 14 or 16 and derivatives thereof, as in Biotin-(CH₂)₄—COO—;haloalkyl-CO—O—, especially trifuoromethylcarbonyl; amino acid acyl or aderivative thereof, as in FmSCH₂CH(NHBOC)CO—O—; arylalkenyl-CO—O—,especially cinnamoyl-CO—O—; alkyl-O—CO—O—, especiallytert-butyl-O—CO—O—; alkenyl-O—CO—O—, especially allyl-O—CO—O—;arylalkyl-O—CO—O—, especially benzyl-O—CO—O—; protecting group as inPO(OBn)₂; most especially R₄ is OH, acyloxy as cinnamoyloxy.

X¹ is H or alkyl (alkyl being 1 to 6 carbon atoms) and R₅ is mostespecially H or C₁ to C₃ alkyl.

The process which comprises providing an ecteinascidin with asubstitutent at the N-12 position, and removing that substituent, istypically carried out using an ecteinascidin compound with an N-12methyl group. Examples of such compounds are to be found in ourpublished and copending PCT patent applications relating toecteinascidin compounds. These applications are incorporated herein infull by specific reference. Removal of the N-12 methyl group can beachieved using known demethylation procedures.

Within the generality of this reaction we also include the process wherethe substituent is a protecting group, and the protecting group isremoved.

The N—H group at the 12-position can be derivatised with a groupR12^(a). Suitable examples are as defined for R^(d) with the exceptionof hydrogen. Preferred examples include acyl, especially alkyl-CO—;alkenyl, especially allyl; or alkyl-O—CO— especially tBOC.

The process for making ecteinascidin 729 and related compounds can bemodelled on the synthetic procedures given in WO 0187895. Typically, theprocess can employ a starting compound with a methyl group at N-12,which is replaced by a protecting group.

For example, in accordance with a process of this invention, a compoundof formula (B):

where R²¹ is as defined, and Prot³ is a protecting group, is subjectedto converting —CH₂NH₂ at the 1-position to —CH₂OH, protecting the —CH₂OHat the 1-position, protecting the —OH at the 18-position, removing themethyl group at the 12-position, deprotecting the 5-position, forming a10-hydroxy-di-6,8-enone, protecting the 12-position, removing theprotecting group at the 1-position, forming a labile group at the1-position set up for giving a 1,4 bridge, forming the 1,4 bridge,deprotecting the 18-position, optionally modifying the 1,4 bridge,removing the protecting group at the 12-position, and optionally furthermodifying the structure.

The —CH₂OH at the 1-position is protected for example with atert-butyldiphenylsilyloxy group. The —OH at the 18-position isprotected for example with a methoxyethoxymethyl group. The 12-positionis protected for example with an allyl group. A labile group at the1-position is typically formed using a reagent of formula:—CH₂—X′—C(═O)—CHNProt²-SH,where X′ is —O — or —NProt¹-, and Prot¹ and Prot² are as defined. The1,4 bridge can be modified, as for example by removing protection from—NProt¹- to give —NH— which may then be further derivatised,deprotecting —NProt²- to —NH— and optionally converting to —C(═O)— whichmay then be further derivatised, thus giving the range of possibilitiesfor the 1,4 bridge defined by the given formula—⁽¹⁾CH₂—X—C(═O)—C(R^(a))(R^(b))—S⁽⁴⁾—.

Examples of further modifications which may be carried out on thestructure include one or more of altering the 1-substituent such asconverting a 21-nitrile group to a 21-hydroxy group altering the5-substituent, altering the 18-substituent, oxidising the sulphur in the1,4 bridge, adding a substituent at the 12-position, and converting ringE to a quinone.

FURTHER DETAILS OF THE INVENTION

Intermediates 1 and 16 (denoted as intermediates 21 and 36 respectivelyin patent applications WO 0069862 and WO 0187895) are useful for thepreparation of other ecteinascidin compounds as detailed herein. Alsoother quinone related analogues are described herein from intermediate16 and 18 (denoted as intermediate 35 in patent applications WO 0069862and WO 0187895).

In particular, from intermediate 1 it is possible to synthesize thenaturally occurring ecteinascidin compound ET729.

Similarly, from intermediate 16 it is possible to synthesize thenaturally occurring compounds ET594, ET745 and ET759B (via ET770 andET743 as intermediates)) and the quinones related to ET594 and ET736,respectively. The quinone related to ET637 is obtained from intermediate18.

The synthesis of ET729 is described herein; the current invention isalso directed at the preparation of new analogues of ET729 fromintermediate 12 following a similar synthetic sequence.

In a further aspect of the current invention, intermediate 1 is used inthe synthesis of a new family of ecteinascidin analogues (such as 77) inwhich the 1,4 bridge includes an amide linkage rather than the lactonelinkage found in ET-743.

Thus according to the present invention, we provide ecteinascidinderivatives having an amide linkage rather than the lactone linkagefound in ET-743, or lacking the bridge. The definition of the newcompounds excludes the known ecteinascidin compounds. The new compoundsinclude those in the table at the end of this specification, andanalogues thereof. The analogues may differ by one or more substituentsfrom those exemplified in our WO0187894, WO0187895 or WO0069862, andgenerally are within the relevant formulae given in our WO0187894,WO0187895 or WO0069862.

Thus, a general formula for the compounds of this invention is arrivedat by identifying a new compound in the present specification,especially the table, and generalising in accordance with thedefinitions of the rest of the molecule based on a general formula ofthe preceding applications. Preferred definitions given in our earlierWO filings will then also apply.

The synthetic methods of the present invention provide the first methodsfor the preparation of ET729, ET594, ET745 and ET759B; and the quinonesrelated of ET594, ET637, ET736 and the bridged lactam analog of ET743and related intermediates. Furthermore the present invention providesthe first synthetic methods for preparation of different analogs ofET729.

Such synthetic routes may provide more economic paths to the knownantitumour agents, as well as permitting preparation of new activecompounds.

Suitable starting materials for the new synthetic processes includecompounds related to the natural bis(tetrahydroisoquinoline) alkaloids.Such starting materials may be prepared either from the differentclasses of saframycin and safracin antibiotics available from differentculture broths as detailed in patent applications WO 0187894 and WO0187895 or by other synthetic or biochemical processes.

In one particular aspect, the present invention is directed at the useof the Intermediate 1 (intermediate 21 in patent applications WO 0187894and WO 0187895) in a new synthetic process for the preparation ofecteinascidin 729 as detailed in Scheme I.

In general, the conversion of Intermediate 1, or a related compound, toan ecteinascidin product such as ET729 involves the followingtransformations:

-   (a) Conversion of the NH₂ to OH by reaction, for example with sodium    nitrite in acetic acid.-   (b) Protection of the primary OH and the E-ring phenol.-   (c) Demethylation of the bridged secondary amine followed by    deprotection and oxidation of the A-ring phenol and subsequent    allylation of the bridged amine.-   (d) Deprotection and esterification of the primary alcohol with a    protected cysteine sidechain to give intermediate 10.-   (e) Creation of the bridged ring by cyclization reaction (to    give 11) and subsequent N and O deprotection reactions to give    intermediate 12.-   (f) Introduction of the dopamine residue by transamination and    Pictect-Spengler reactions to give intermediate 14.-   (g) Removal of the N allyl protecting group and conversion of the CN    to OH.

Therefore in summary, it is now feasible to transform intermediate 1(obtainable from cyanosafracin B) into ET-729, resulting in the firstsynthetic approach to this naturally occurring ecteinascidin.

The high functionality of the intermediate compounds necessitates theuse of protecting groups for the E-ring phenol, the cysteine sidechain,the bridged nitrogen and the primary alcohol in order to preventunwanted side reactions.

As such, a number of alternative intermediates can be generateddependent on the particular selection of protecting groups. The use ofother protecting group strategies not detailed is also part of thisinvention.

In a further aspect, the current invention provides new processes forthe conversion of intermediate 16 (denoted as intermediate 36 in patentapplications WO 0069862 and WO 0187895) into the naturally occurringecteinascidin compounds ET594, ET745 and ET759B as detailed in Scheme 2.

Intermediate 16 is obtained from intermediate 1 as described in theabove mentioned patent applications, and has the same structure ofintermediate 13 of scheme 1 with -Me bonded to N instead of -Allyl.

In more detail such processes involve the following conversions.

-   (a) Generation of ET594 from intermediate 16 in a single step by    conversion of the CN group into an OH group.-   (b) Synthesis of ET745 from ET743 (obtained in two steps from    intermediate 16) by reductive cleavage of the secondary alcohol    functionality.-   (c) Generation of ET759B from intermediate 16 in three steps    involving the formation of ET770 followed by oxidation and    conversion of the nitrile group into a hydroxy group.

Thus, the current invention also provides simple new methods forproducing the naturally occurring ecteinascidin compounds ET594, ET745and ET759B from intermediate 16 (obtainable from cyanosafracin B).

Furthermore, the current invention provides a process for the synthesisof the quinone derivatives of ET594, ET637 and ET736 from intermediate16 (denoted as intermediate 36 in patent applications WO 0187894 and WO0187895) and intermediate 18 (denoted as intermediate 35 in patentapplications WO 0069862 and WO 0187895). (Scheme 3).

In more detail, such processes involve the following conversions.

-   (a) Generation of ET637-quinone from intermediate 18 in three steps    involving the formation of intermediate 19 by acetylation of the    amine group; followed by oxidation of the phenolic ring and    conversion of the nitrile group into a hydroxyl group.-   (b) Synthesis of ET594-quinone in two steps from intermediate 16    through an oxidation reaction of the phenolic ring and conversion of    the nitrile group into a hydroxyl group.-   (c) Synthesis of ET736-quinone in three steps from intermediate 16    involving the introduction of the tryptamine moiety to generate    intermediate 22, oxidation reaction and conversion of the nitrile    group into a hydroxyl group.

Thus, the current invention provides short and new methods for producingthe oxidized derivatives of the naturally occurring ecteinascidincompounds Et594, ET637 and ET736 from intermediates 16 and 18 (bothobtainable from cyanosafracin B).

In a further embodiment and following the synthetic sequence of ET729,the present invention provides processes for producing new and differentanalogues of ET729 from intermediate 12. The preferred methods ofproducing the compounds of formula I, II and III are described below inthe following reaction schemes with examples of typical substituentgroups.

In general, the conversion of Intermediate 12 or 13, to differentanalogues of ET729 involves the following transformations:

-   (a) Acylation reactions through the different procedures described    in, the experimental part, deallylation reactions at N-12 and    interconversion of the nitrile group into the hydroxyl group.    Compound 40 is an example of intermediate with typical substituent    groups wherein two consecutive acylation reactions has placed,    followed by the two last steps described above.-   (b) Generation of compound 35 form intermediate 30 in a single step    by acetylation reaction at N-12.-   (c) Synthesis of compound 43 from compound 13 by deallylation    reaction and conversion of the nitrile group into the hydroxyl group    with CuCl.

In more detail, such processes involve the following transformationsfrom intermediate 13.

-   (a) Generation of intermediate 46 in two steps from compound 14 by    protection of the hydroxyl group as Boc carbonate and deacetyllation    at C-5 with KOH.-   (b) Synthesis of compounds 53 and 54 from intermediate 46 following    the same synthetic sequence: Acylation reaction at C-5 with    cinnamoyl chloride or octanoic acid, deprotection of the carbonate    group, deallylation reaction at N-12 and finally conversion of the    nitrile group into the hydroxyl group.-   (c) Synthesis of different analogues of ET729 from intermediate 13    involving three steps: introduction of the triptamine moiety,    deallylation reaction and conversion of the nitrile group into the    hydroxyl group.

In more detail, scheme 6 describes the synthesis of compound 67 and 68from intermediate 14 and ET729 respectively.

In a further embodiment, the current invention provides a process forthe synthesis of a new family of ecteinascidin compounds wherein thelactone linkage of the 1,4 bridge of ET743 and related intermediates isreplaced with an amide linkage, as detailed in Scheme 7.

In more detail, intermediate 1 (intermediate 21 in patent applicationsWO 0069862 and WO 0187895) can be converted to such compounds throughthe following sequence of steps:

-   (a) Introduction of a protected cysteine fragment in a single step    by coupling with the primary amine functionality of intermediate 1.-   (b). Protecting group manipulations and an oxidation reaction to    generate intermediate 72.-   (c) Cyclization to generate the desired lactam bridge structure    followed by deprotection of the primary amine.-   (d) Completion of the synthesis through transamination,    Pictect-Spengler reaction and conversion of the nitrile into a    hydroxyl group.

Thus this invention provides a process for the synthesis of a largefamily of compounds related to ET743 in which the lactone linkage of thebridge structure has been replaced by a lactam linkage.

As the skilled artisan will readily appreciate, the reaction schemesdescribed herein may be modified and/or combined in various ways, andthe alternative sequences of steps and the compounds generated therefromare part of this invention.

Thus, by these and other routes, it is possible to transformcyanosafracin B into a number of intermediates and derivatives withpotential antitumor therapeutic activity. These intermediates can bemade starting from already described compounds, or using alternativeroutes.

Novel Active Compounds

We have additionally found that certain of the compounds of theinvention which we initially prepared as intermediates have exceptionalactivity in the treatment of cancers, such as leukaemias, lung cancer,colon cancer, kidney cancer and melanoma.

Thus, the present invention provides a method of treating any mammal,notably a human, affected by cancer which comprises administering to theaffected individual a therapeutically effective amount of a compound ofthe invention, or a pharmaceutical composition thereof.

The present invention also relates to pharmaceutical preparations, whichcontain as active ingredient a compound or compounds of the invention,as well as the processes for their preparation.

Examples of pharmaceutical compositions include any solid (tablets,pills, capsules, granules, etc.) or liquid (solutions, suspensions oremulsions) with suitable composition or oral, topical or parenteraladministration, and they may contain the pure compound or in combinationwith any carrier or other pharmacologically active compounds. Thesecompositions may need to be sterile when administered parenterally.

Administration of the compounds or compositions of the present inventionmay be by any suitable method, such as intravenous infusion, oralpreparations, intraperitoneal and intravenous administration. We preferthat infusion times of up to 24 hours are used, more preferably 2-12hours, with 2-6 hours most preferred. Short infusion times which allowtreatment to be carried out without an overnight stay in hospital areespecially desirable. However, infusion may be 12 to 24 hours or evenlonger if required. Infusion may be carried out at suitable intervals ofsay 2 to 4 weeks. Pharmaceutical compositions containing compounds ofthe invention may be delivered by liposome or nanosphere encapsulation,in sustained release formulations or by other standard delivery means.

The correct dosage of the compounds will vary according to theparticular formulation, the mode of application, and the particularsitus, host and tumour being treated. Other factors like age, bodyweight, sex, diet, time of administration, rate of excretion, conditionof the host, drug combinations, reaction sensitivities and severity ofthe disease shall be taken into account. Administration can be carriedout continuously or periodically within the maximum tolerated dose.

The compounds and compositions of this invention may be used with otherdrugs to provide a combination therapy. The other drugs may form part ofthe same composition, or be provided as a separate composition foradministration at the same time or a different time. The identity of theother drug is not particularly limited, and suitable candidates include:

a) drugs with antimitotic effects, especially those which targetcytoskeletal elements, including microtubule modulators such as taxanedrugs (such as taxol, paclitaxel, taxotere, docetaxel), podophylotoxinsor vinca alkaloids (vincristine, vinblastine);

b) antimetabolite drugs such as 5-fluorouracil, cytarabine, gemcitabine,purine analogues such as pentostatin, methotrexate);

c) alkylating agents such as nitrogen mustards (such as cyclophosphamideor ifosphamide);

d) drugs which target DNA such as the antracycline drugs adriamycin,doxorubicin, pharmorubicin or epirubicin;

e) drugs which target topoisomerases such as etoposide;

f) hormones and hormone agonists or antagonists such as estrogens,antiestrogens (tamoxifen and related compounds) and androgens,flutamide, leuprorelin, goserelin, cyprotrone or octreotide;

g) drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin;

h) alkylating drugs such as platinum drugs (cis-platin, carbonplatin,oxaliplatin, paraplatin) or nitrosoureas;

i) drugs potentially affecting metastasis of tumours such as matrixmetalloproteinase inhibitors;

j) gene therapy and antisense agents;

k) antibody therapeutics;

l) other bioactive compounds of marine origin, notably the didemninssuch as aplidine;

m) steroid analogues, in particular dexamethasone;

n) anti-inflammatory drugs, in particular dexamethasone; and

o) anti-emetic drugs, in particular dexamethasone.

The present invention also extends to the compounds of the invention foruse in a method of treatment, and to the use of the compounds in thepreparation of a composition for treatment of cancer.

EXAMPLES

The present invention is illustrated by the following examples.

Experimental Part

Example 1

A solution of 1 (9.84 g, 18.97 mmol) in THF (569 mL) and H₂O (285 mL)was cooled at OC with an ice bath. Then, NaNO₂ (1.96 g, 28.45 mmol) and90% aq. AcOH (18.97 mL, 0.33 mol) were added at 0° C. and the mixturewas stirred at 23° C. for 18 h. After cooling down the reaction to 0°C., a saturated aqueous sodium bicarbonate solution (300 mL, basic pH)and dichloromethane (500 mL) were added. After extraction, the aqueousphase was further extracted with dichloromethane (2×300 mL). Thecombined organic extracts were dried over sodium sulphate and evaporatedto dryness under reduced pressure. The crude solid was then dissolved inMeOH (379 mL), and 1M NaOH (38 mL) was added at 0° C. The mixture wasstirred at 23° C. for 4 h. After dilution with EtOAc (600 mL) at 0° C.,the organic layer was washed with a mixture of water (400 mL) and, asaturated aqueous sodium bicarbonate solution (100 mL, basic pH). Afterextraction, the aqueous phase was further extracted with EtOAc (3×300mL). The combined organic extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by flash columnchromatography (SiO₂, Hex:EtOAc gradient from 3:1 to 2:1) to afford 2(4.55 g, 46%) as a white solid.

R_(f): 0.33 (Hex:EtOAc 1:1).

¹H NMR (300 MHz, CDCl₃) 6.48 (s, 1H), 6.15-6.02 (m, 1H), 5.92 (d, 1H),5.86 (d, 1H), 5.77 (s, 1H), 5.39 (dd, 1H), 5.26 (dd, 1H), 4.24-4.15 (m,3H), 4.04 (d, 1H), 3.97 (t, 1H), 3.74 (s, 3H), 3.64 (dt, 1H), 3.43 (dd,1H), 3.38-3.34 (m, 2H), 3.31 (t, 1H), 3.22 (dd, 1H), 3.10 (dd, 1H), 2.49(d, 1H), 2.34 (s, 3H), 2.24 (s, 3H), 2.11 (s, 3H), 1.88 (dd, 1H).

¹³C NMR (75 MHz, CDCl₃) 148.6, 146.7, 144.4, 143.0, 138.9, 133.9, 130.2,129.1, 121.1, 120.9, 117.7, 117.4, 116.8, 113.3, 112.3, 101.1, 74.3,63.7, 60.6, 60.1, 58.1, 56.9, 56.7, 55.4, 41.7, 26.2, 25.7, 15.7, 9.3.

ESI-MS m/z: Calcd. for C₂₉H₃₃N₃O₆: 519.59. Found (M+1)⁺: 520.3.

Example 2

To a solution of 2 (9.33 g, 0.018 mol), in anhydrous DMF (40 mL, 0.45 M)was added at 23° C. imidazole (3.05 g, 0.045 mol) and DMAP (219 mg,0.0018 mol). The solution was cooled at 0° C. and TBDPSCl (7.0 mL, 0.027mol) was dropwise added under argon atmosphere. The reaction mixture wasallowed to reach 23° C. and left at this temperature for 1 hour and 15minutes. After this time, water (350 mL) and a mixture of ethylacetate/hexane (3:2, 250 mL) were added. The organic phase wasseparated, dried over sodium sulphate and filtered and the solvent waseliminated under reduced pressure. The crude material was purified byflash column chromatography (eluent mixtures of ethyl acetate/hexane ingradient from 15:85 to 2:3) to afford 3 (11.8 g, 87%) as a yellow solid.

R_(f): 0.36 (ethyl acetate/hexane 2:3)

¹H-RMN (300 MHz, CDCl₃): δ 7.55 (d, 2H), 7.41-7.22 (m, 8H), 6.45 (s,1H), 6.18-6.02 (m, 1H), 6.78 (s, 2H), 6.61 (s, 1H), 5.35 (d, 1H), 5.21(d, 1H), 4.42 (d, 1H), 4.18 (m, 2H), 4.05 (m, 2H), 3.78 (s, 3H), 3.64(dd, 1H), 3.41-3.31 (m, 2H), 3.29-3.20 (m, 2H); 3.02 (dd, 1H), 2.70 (d,1H), 2.30 (s, 3H), 2.28 (s, 3H), 2.08 (s, 3H), 2.02 (dd, 1H), 0.90 (s,9H).

¹³C-RMN (75 MHz, CDCl₃): δ 148.69, 146.94, 144.33, 142.90, 139.35,135.98, 135.67, 134.27, 133.56, 132.94, 131.49, 129.84, 129.70, 128.59,127.79, 127.73, 122.13, 121.24, 118.94, 117.61, 117.55, 113.22, 112.04,101.12, 74.52, 68.24, 61.89, 60.93, 59.29, 57.68, 57.06, 55.73, 42.01,26.93, 26.79, 25.84, 19.19, 16.07, 9.56.

ESI-MS m/z: Calcd. for C₄₅H₅₁N₃O₆Si: 757.3. Found (M+Na)⁺: 780.3.

Example 3

To a solution of intermediate 3 (11.75 g, 0.016 mol) in THF/H₂O (113mL/0.31 mL, 0.14 M) was added MEM-chloride (3.0 mL, 0.026 mol). Thesolution was cooled at 0° C. and sodium hydride (930 mg, 0.023 mol) wasportionwise added (1 hour and 15 minutes for the addition). The reactionmixture was left at 0° C. under Argon atmosphere for 1 hour. After thistime water (150 mL) was added and the aqueous phase was extracted withdichloromethane (2×150 mL). The combined organic layers were dried oversodium sulphate, filtered and the solvent was eliminated under reducedpressure to afford intermediate 4 (13.4 g, 100%) as a yellow solid. Thiscompound is used for the next step without purification.

R_(f): 0.32 (ethyl acetate/hexane 1:2)

¹H-RMN (300 MHz, CDCl₃): δ 7.58 (d, 2H), 7.38 (m, 6H), 7.27 (m, 2H),6.70 (s, 1H), 6.18-6.02 (m, 1H), 5.75 (s, 1H), 5.60 (s, 1H), 5.40 (d,1H), 5.28 (d, 1H), 5.24 (d, 1H), 5.19 (d, 1H), 4.50 (broad s, 1H), 4.38(broad s, 1H), 4.20-3.97 (m, 4H), 3.85 (m, 1H), 3.70 (s, 3H), 3.58 (m,3H), 3.38 (s, 3H), 3.38 (m, 2H), 3.22 (m, 2H), 3.02 (dd, 1H), 2.70 (d,1H), 2.38 (s, 3H), 2.24 (s, 3H), 2.05 (s, 3H), 1.93 (dd, 1H), 0.84 (s,9H).

¹³C-RMN (75 MHz, CDCl₃): δ 148.76, 148.60, 148.49, 139.29, 135.87,135.63, 134.18, 133.23, 132.89, 130.85, 130.49, 129.89, 129.77, 127.82,127.73, 125.45, 121.76, 118.45, 117.55, 113.23, 111.97, 101.13, 98.49,95.79, 74.26, 71.98, 71.95, 69.57, 67.44, 67.37, 66.95, 61.32, 59.85,59.18, 59.10, 57.50, 57.16, 55.58, 41.69, 29.87, 26.83, 26.83, 26.12,19.05, 16.07, 9.46.

ESI-MS m/z: Calcd. for C₄₉H₅₉N₃O₈Si: 845.4. Found (M+1)⁺: 846.3.

Example 4

To a solution of intermediate 4 (2.51 g, 0.003 mol) in anhydrousdichloromethane (25 mL, 0.12 M) was added at −20° C. under Argonatmosphere m-CPBA (1.33 g, 0.006 mol). The solution was allowed to reach−10° C. for 25 minutes, TEA (4.14 mL, 0.03 mol) was added and thereaction mixture was left at 0° C., finally TFAA (6.29 mL, 0.045 mol)was dropwise added and the solution kept at 0° C. for 30 minutes. Afterthis time water was added and the aqueous phase was separated, driedover sodium sulphate, filtered and the solvent was eliminated underreduced pressure. The crude was purified by flash column chromatography(eluent mixtures of ethyl acetate/hexane in gradients from 1:4 to 6:1and final washes with methanol) to afford intermediate 5 (2.1 g, 85%) asa yellow solid.

R_(f): 0.19 (ethyl acetate/hexane 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.55 (d, 2H), 7.45-7.28 (m, 8H), 6.70 (s,1H), 6.14-6.02 (m, 1H), 5.81 (d, 1), 5.67 (d, 1H), 5.43-5.35 (m, 2H),5.26 (m, 2H), 5.03 broad s, 1H), 4.73 broad s, 1H), 4.68 (m, 1H),4.22-4.09 (m, 3H), 3.81 (broad s, 2H), 3.73 (s, 3H), 3.61 (dd, 1H), 3.53(broad s, 4H), 3.46-3.28 (m, 2H), 3.34 (s, 3H), 2.97 (d, 1H), 2.25 (s,3H), 2.11 (s, 3H), 1.95 (dd, 1H), 0.94 (s, 9H).

ESI-MS m/z: Calcd. for C₄₈H₅₇N₃O₈Si: 831.4. Found (M+Na)⁺: 832.3.

Example 5

To a solution of intermediate 5 (5.9 g, 7.09 mmol), (PPh₃)₂PdCl₂ (399mg, 0.57 mmol), acetic acid (2.03 mL, 35.47 mol) in anhydrousdichloromethane (45 mL, 0.16 M) was dropwise added at 23° C. tributyltinhydride (6.7 mL, 24.83 mmol). The reaction mixture was left at 23° C.and under Argon atmosphere for 35 minutes. The reaction mixture waspoured onto column (eluent mixtures of ethyl acetate/hexane in gradientfrom 1:4 to 8: 1) to afford intermediate 6 (3.97 g, 71%) as a yellowsolid.

R_(f): 0.17 (ethyl acetate/hexane 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.57(d, 2H), 7.43-7.24 (m, 8H), 6.68 (s, 1H),5.73 (d, 1H), 5.58 (d, 1H), 5.47 (d, 1H), 5.22 (d, 1H), 4.54 (d, 1H),4.47 (d, 1H), 4.14 (m, 1H), 4.04 (dd, 1H), 3.94 (m, 1H), 3.73-3.65 (m,4H), 3.70 (s, 3H), 3.39 (s, 3H), 3.38-3.30 (m, 2H), 3.25 (m, 1H), 3.11(dd, 1H); 2.91 (d, 1H), 2.24 (s, 3H), 2.06 (s, 3H), 1.84 (dd, 1H), 0.91(s, 9H).

ESI-MS m/z: Calcd. for C₄₅H₅₃N₃O₈Si: 791.4. Found (M+Na)⁺: 814.3.

Example 6

To a solution of intermediate 6 (1.87 g, 2.36 mmol) in anhydrousdichloromethane (20 mL, 0.12 M) was dropwise added at −15 PC under Argonatmosphere a solution of benceneseleninic anhydride (1.82 g, 3.53 mmol)in anhydrous dichloromethane (20 mL). The solution was left at −15° C.for 25 minutes. The reaction mixture was diluted with dichloromethane,and a saturated solution of sodium bicarbonate was added at −10° C. Theaqueous phase was extracted with dichloromethane, the combined organiclayers were dried over sodium sulphate, filtered and the solvent waseliminated under reduced pressure. The crude of the reaction waspurified by flash column chromatography (eluent mixtures of ethylacetate/hexane in gradient from 1:6 to 6:1) to afford intermediate 7(1.53 g, 80%) as a yellow solid and as a mixture of isomers 3:1 by¹H-RMN.

R_(f): 0.24 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.64 (dd, 6H), 7.57 (d, 2H), 7.40-7.25 (m,12H), 6.65 (s, 1H), 6.53 (s, 1H), 5.65 (s, 1H), 5.42 (s, 1H), 5.25 (s,1H), 5.23 (s, 1H), 5.22 (m, 1H), 5.19 (d, 1H), 5.11 (d, 1H), 5.06 (d,1H), 4.80 (s, 1H), 4.73 (s, 1H), 4.43 (d, 1H), 4.36 (m, 1H), 4.32 (m,1H), 4.25 (d, 1H), 3.97 (dd, 1H); 3.89 (s, 3H), 3.86-3.77 (m, 4H),3.74-3.60 (m, 4H), 3.59 (s, 3H), 3.55-3.48 (m, 4H), 3.38-3.35 (m, 2H),3.34 (s, 3H), 3.31 (s, 3H), 3.18-3.03 (m, 2H), 2.96 (dd, 1H), 2.73 (d,1H), 2.57 (d, 1H), 2.21 (s, 3H), 2.14 (s, 3H), 2.17-1.86 (m, 2H), 1.75(s, 3H), 1.70 (s, 3H), 1.07 (s, 9H), 0.99 (s, 9H).

¹³C-RMN (75 MHz, CDCl₃): δ 201.05, 197.78, 160.43, 158.64, 148.81,147.84, 146.88, 146.70, 140.01, 137.97, 135.99, 135.97, 135.79, 133.64,133.00, 132.80, 131.33, 131.25, 130.61, 130.41, 130.05, 129.98, 129.91,129.04, 127.95, 127.91, 127.77, 127.60, 125.98, 125.82, 117.38, 117.26,113.51, 111.22, 104.50, 104.37, 101.39, 100.55, 98.16, 95.84, 92.51,73.09, 71.96, 71.94, 71.92, 70.48, 69.67, 69.60, 67.65, 66.99, 64.65,60.68, 60.23. 60.12, 60.02, 59.35, 59.26, 59.24, 59.22, 59.19, 59.03,56.81, 56.44, 50.30, 49.99, 49.73, 49.61, 43.24, 36.30, 31.30, 27.10,19.57, 19.23, 16.03, 16.01, 7.55, 7.27.

ESI-MS m/z: Calcd. for C₄₅H₅₃N₃O₉Si: 807.4. Found (M+1)⁺: 808.3.

Example 7

To a solution of intermediate 7 (3.78 g, 4.68 mmol) in anhydrous DMF (30mL, 0.16 M) was added at 23° C. and under Argon atmosphere cesiumcarbonate (5.35 g, 16.39 mmol) and allyl bromide (2.03 mL, 23.42 mmol).The reaction mixture was left at 23° C. for 16 hours, cooled at 0° C.and dropwise added acetic acid to destroy the excess of base. Thesolution was diluted with dichloromethane and a saturated solution ofsodium bicarbonate was dropwise added. The aqueous phase was extractedwith dichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentmixtures of hexane/ethyl acetate in gradient from 100/0 to 2:1) toafford intermediate 8 (3.62 g, 91%) as a yellow solid.

R_(f): 0.40 (ethyl acetate/hexane 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.65 (m, 3H), 7.43-7.28 (m, 7H), 6.54 (s,1H), 5.88 (m, 1H), 5.31-5.10 (m, 2H), 5.24 (s, 1H), 5.19 (s, 1H), 5.12(d, 1H), 5.02 (d, 1H), 4.47 (d, 1H), 4.34 (dd, 1H), 3.99 (dd, 1H),3.93-3.86 (m, 2H), 3.81-3.73 (m, 2H), 3.62-3.53 (m, 2H), 3.61 (s, 3H),3.41 (m, 1H), 3.37 (s, 3H), 3.23 (m, 1H), 3.08-2.97 (m, 2H), 2.79 (ddd,2H), 2.39 (d, 1H), 2.24-1.95 (m, 1H), 2.17 (s, 3H), 1.71 (s, 3H), 1.07(s, 9H).

ESI-MS m/z: Calcd. for C₄₈H₅₇N₃O₉Si: 847.4. Found (M+1)⁺: 848.2.

Example 8

To a solution of intermediate 8 (942 mg, 1.11 mmol) in anhydrous THF (10mL, 0.1 M) and under Argon atmosphere was dropwise added TBAF (3.33 mL,3.33 mmol) at 23° C. The reaction mixture was left at 23° C. under Argonatmosphere for 2 hours and 20 minutes. The solution was diluted withethyl acetate and a saturated solution of brine was added. The organicphase was separated, dried over sodium sulphate, filtered and thesolvent was eliminated under reduced pressure. The crude was purified byflash column chromatography (eluent mixtures of ethyl acetate/hexane ingradient from 1:2 to 2:1) to afford intermediate 9 (461 mg, 68%) as ayellow solid.

R_(f): 0.26 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.66 (s, 1H), 6.60 (s, 1H), 5.88-5.80 (m,1H), 5.81 (s, 3H), 5.80 (s, 3H), 5.20 (d, 2H), 5.12 (d, 1H), 5.07 (d,1H), 4.12 (m, 1H), 4.07 (m, 1H), 3.91-3.67 (m, 4H), 3.85 (s, 3H),3.59-3.49 (m, 4H), 3.41 (broad d, 1H), 3.34 (m, 1H), 3.31 (s, 3H), 3.24(dt, 1H), 3.09 (dd, 1H), 2.86 (ddd, 2H), 2.53 (d, 2H), 2.18 (s, 3H),2.05 (d, 2H), 1.75 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 198.81, 159.04, 149.01, 148.23, 140.94,135.34, 131.42, 130.99, 125.74, 123.58, 117.99, 117.21, 111.06, 104.41,101.86, 98.52, 71.86, 70.57, 69.43, 62.25, 60.63, 59.22, 59.19, 58.46,56.68, 56.22, 55.74, 51.89, 36.57, 25.79, 15.98, 7.52.

ESI-MS m/z: Calcd. for C₃₂H₃₉N₃O₉: 609.3. Found (M+Na)⁺: 632.3.

Example 9

To a solution of intermediate 9 (1.43 g, 2.34 mmol) and cysteinederivative (1.40 g, 3.51 mmol) in anhydrous dichloromethane (20 mL, 0.12M), was added at 23° C. EDC.HCl (1.12 g, 5.85 mmol), DMAP (144 mg, 1.17mmol) and DIPEA (0.24 mL, 1.36 mmol). The reaction mixture was leftunder Argon atmosphere for 2 hours. A saturated solution of sodiumbicarbonate was added and the aqueous phase was extracted withdichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentmixtures of ethyl acetate/hexane in gradient from 1:4 to 2:1) to affordintermediate 10 (1.42 g, 61%, some starting material was recuperated) asa yellow solid and as a mixture of 4 isomers.

R_(f): 0.26 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.70 (d, 8H), 7.66-7.58 (m, 8H), 7.37-7.23(m, 16H), 6.59 (broad s, 2H), 6.49 (s, 1H), 6.47 (s, 1H), 5.87-5.79 (m,4H), 5.69, 5.67, 5.65 (broad s, 6H), 5.56 (s, 2H), 5.38-4.97 (m, 20H),4.61-4.37 (m, 12H), 4.18-3.85 (m, 28H), 3.78 (s, 3H), 3.77-3.66 (m, 2H),3.59 (s, 3H), 3.58 (s, 3H), 3.58 (s, 3H), 3.53 (m, 8H), 3.35 (m, 2H),3.35 (s, 3H), 3.35 (s, 3H), 3.32 (s, 3H), 3.31 (s, 3H), 3.21-2.55 (m,36H), 2.43-2.30 (m, 4H), 2.17 (s, 6H), 2.12 (s, 3H), 2.10 (s, 3H), 1.74,1.74, 1.73 (s, 12H), 1.22 (s, 36H).

ESI-MS m/z: Calcd. for C₅₄H₆₂N₄O₁₂S: 990.4. Found (M+1)⁺: 991.2.

Example 10

The reaction flask was flamed twice, purged vacuum/Argon several timesand kept under Argon atmosphere for the reaction. To a solution of DMSO(43.0 μl) in anhydrous CH₂Cl₂ (4.0 mL) was dropwise added triflicanhydride (20.3 μL) at −78° C. The reaction mixture was stirred at −78°C. for 20 minutes, then a solution of 10 (major isomer) (60 mg, 0.06mmol) in anhydrous CH₂Cl₂ (2.0 mL) at −78° C. was added via canula.During the addition the temperature was kept at −78° C. in both flasks.The reaction mixture was stirred at −40° C. for 35 minutes. After thistime, ^(i)Pr₂Net (160 μL) was dropwise added and the reaction mixturewas kept at 0° C. for 45 minutes. Then ^(t)BuOH (57 μL) and guanidine(96 μL) were dropwise added and the reaction mixture was stirred at 23°C. for 40 minutes. After this time, acetic anhydride (86 μL), wasdropwise added and the reaction mixture was kept at 23° C. for 1 hourmore. Then the reaction mixture was diluted with CH₂Cl₂ and washed withan aqueous saturated solution of NH₄Cl, NaHCO₃ and NaCl. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated.

The residue was purified by a flash column chromatography (eluentmixtures of ethyl acetate/hexane in gradient from 1:4 to 1:1) to afford11 (34 mg, 67%) as a pale yellow solid.

R_(f): 0.43 (ethyl acetate/hexane 1:1)

1H-RMN (300 MHz, CDCl₃): δ 6.79 (s, 1H), 6.08 (d, 1H), 5.98 (d, 1H),5.92-5.82 (m, 1H), 5.32 (d, 1H), 5.18 (m, 1H), 5.17 (d, 1H), 5.12 (d,1H), 5.01 (d, 1H), 4.62 (d, 1H), 4.52 (broad s, 1H), 4.41 (d, 1H), 4.28(m, 2H), 4.20 (d, 1H), 4.14 (dd, 1H), 3.91 (oct, 2H), 3.76 (s, 3H), 3.59(t, 2H), 3.54 (m, 1H), 3.44 (d, 1H), 3.37 (s, 3H), 3.00 (m, 2H),2.90-2.72 (m, 3H), 2.37-2.24 (m, 1H), 2.31 (s, 3H), 2.29 (s, 3H), 2.02(s, 3H), 1.45 (s, 9H).

¹³C-RMN (75 MHz, CDCl₃): δ 170.89, 168.86, 155.36, 149.40, 148.60,145.97, 141.19, 140.64, 135.47, 131.65, 131.15, 125.54, 125.24, 120.68,118.18, 118.09, 113.78, 113.54, 102.21, 98.33, 79.95, 71.93, 69.35,61.60, 60.60, 60.44, 59.93, 59.39, 59.30, 55.90, 54.14, 54.03, 51.18,41.95, 33.06, 28.72, 28.45, 23.93, 20.59, 16.11, 14.42, 9.83.

ESI-MS m/z: Calcd. for C₄₂H₅₂N₄O₁₂S: 836.3. Found (M+1)⁺: 837.1

Example 11

To a solution of intermediate 11 (29 mg, 0.035 mmol) in CHCl₃ (1 mL,0.03 M) was added at 23° C. p-TsOH (40 mg, 0.21 mmol). The reactionmixture was left at 23° C. and under Argon atmosphere for 15 hours. Thereaction was diluted with dichloromethane and a saturated solution ofsodium bicarbonate was added. The aqueous phase was extracted withdichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentmixtures of ethyl acetate/hexane in gradient from 1:4 to 2:1 and finalwashes with methanol) to afford intermediate 12 (16 mg, 71%) as a yellowsolid.

R_(f): 0.07 (ethyl acetate/hexane 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.52 (s, 1H), 6.07 Id, 1H), 5.98 (d, 1H),5.85 (m, 1H), 5.13-5.06 (m, 2H), 5.01 (d, 1H), 4.52 (broad s, 1H), 4.33(d, 1H), 4.26 (s, 1H), 4.19 (d, 1H), 4.12 (m, 1H), 3.77 (s, 3H), 3.53(broad d, 1H), 3.40 (d, 1H), 3.28 (m, 1H), 2.95-2.75 (m, 4H), 2.30 (s,3H), 2.28 (s, 3H), 2.21 (broad s, 2H), 2.03 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 174.59, 168.91, 147.85, 145.86, 143.10,141.22, 140.56, 135.21, 131.27, 129.53, 121.11, 120.72, 118.93, 118.30,114.01, 113.54, 102.15, 61.55, 60.44, 60.30, 59.73, 59.53, 56.09, 54.22,53.31, 52.07, 41.95, 34.58, 24.23, 20.82, 15.89, 9.86.

ESI-MS m/z: Calcd. for C₃₃H₃₆N₄O₈S: 648.2. Found (M+1)⁺: 649.1

Example 12

To a solution of the pyridinium salt (211 mg, 0.85 mmol) in DMF (2.3 mL)was added at 23° C. a solution of intermediate 12 (55 mg, 0.085 mmol) indichloromethane (2.9 mL, 0.016 M final concentration). The reactionmixture was left at 23° C. and under Argon atmosphere for 4 hours and 15minutes, then DBU (13 μL, 0.085 mmol) was added and the solution wasstirred at 23° C. and under Argon atmosphere for 15 minutes. After thistime a saturated solution of oxalic acid (2 mL) was added, and thereaction mixture was left at 23° C. under Argon atmosphere for 30minutes. The reaction mixture was cooled at 0° C., was diluted with Et₂Oand a saturated solution of sodium bicarbonate was added until to reachpH=5. The aqueous phase was extracted with Et₂O (×4), further basifiedwith more sodium bicarbonate and extracted with more Et₂O (×4). Thecombined organic layers were dried over sodium sulphate, filtered andthe solvent was eliminated under reduced pressure. The crude waspurified by flash column chromatography (eluent mixtures ethylacetate/hexane in gradient from 1:2 to 2:1) to afford intermediate 13(28 mg, 51%) as a yellow solid.

R_(f): 0.66 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.49 (s, 1H), 6.11 (d, 1H), 6.01 (d, 1H),5.88-5.77 (m, 1H), 5.70 (s, 1H), 5.09 (m, 3H), 4.66 (broad s, 1H), 4.40(s, 1H), 4.36 (d, 1H), 4.20 (dd, 1H), 4.17 (d, 1H), 3.75 (s, 3H), 3.55(m, 2H), 2.88-2.67 (m, 5H), 2.56 (d, 1H), 2.32 (s, 3H), 2.25 (s, 3H),2.04 (s, 3H).

ESI-MS m/z: Calcd. for C₃₃H₃₃N₃O₉S: 647.1. Found (M+1)⁺: 648.1

Example 13

To a solution of intermediate 13 (26 mg, 0.04 mmol) and dopaminederivative (24 mg, 0.14 mmol) in EtOH (0.7 mL, 0.06 M) was added at 23°C. silica gel (56 mg). The reaction mixture was left at 23° C. and underArgon atmosphere for 15 hours. The solvent of the reaction waseliminated under reduced pressure and the crude was purified by flashcolumn chromatography (eluent mixtures of ethyl acetate/hexane ingradient from 1:1 to 4:1) to afford intermediate 14 (30 mg, 94%) as apale yellow solid.

R_(f): 0.37 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.60 (s, 1H), 6.46 (s, 1H), 6.44 (s, 1H),6.04 (d, 1H), 5.96 (d, 1H), 5.94-5.80 (m, 1H), 5.73 (s, 1H), 5.48 (broads, 1H), 5.11 (m, 2H), 5.02 (d, 1H), 4.57 (broad s, 1H), 4.36 (d, 1H),4.33 (s, 1H), 4.19 (d, 1H), 4.11 (dd, 1H), 3.78 (s, 3H), 3.61 (s, 3H),3.55 (m, 1H), 3.50 (d, 1H9, 3.09 (m, 1H), 2.99-2.74 (m, 5H), 2.59 (m,1H), 2.47 (dt, 1H), 2.32 (s, 3H), 2.25 (s, 3H), 2.23-2.13 (m, 2H), 2.04(s, 3H).

ESI-MS m/z: Calcd. for C₄₂H₄₄N₄O₁₀S: 796.3. Found (M+1)⁺: 797.2

Example 14

To a solution of intermediate 14 (30 mg, 0.038 mmol), (PPh₃)₂PdCl₂ (3mg, 0.003 mmol), acetic acid (11 μL, 0.188 mmol) in anhydrousdichloromethane (1 mL, 0.04 M) was dropwise added at 23° C. and underArgon atmosphere HSnBu₃ (36 μL, 0.13 mmol). The reaction mixture wasleft at 23° C. under Argon atmosphere for 20 minutes. After this timethe reaction mixture was poured onto column (eluent mixtures ofdichloromethane/methanol in gradient from 100/0 to 30:1 to affordintermediate 15 (12 mg, 42%) as a pale yellow solid. Some startingmaterial (17 mg) was isolated impurified with traces ofbutyltinderivatives.

R_(f): 0.22 (dichloromethane/methanol 20:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.62 (s, 1H), 6.47 (s, 1H), 6.44 (s, 1H),6.06 (d, 1H), 5.98 (d, 1H), 5.03 (d, 1H), 4.57 (broad s, 1H), 4.50 (d,1H), 4.34 (s, 1H), 4.20 (d, 1H), 4.15 (dd, 1H), 3.85 (d, 1H), 3.78 (s,3H), 3.62 (s, 3H), 3.52 (d, 1H), 3.15-2.95 (m, 3H), 2.77 (m, 1H), 2.60(m, 1H), 2.46 (dt, 1H), 2.35 (d, 1H), 2.31 (s, 3H), 2.26 (s, 3H), 2.15(d, 1H), 2.04 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 172.84, 172.12, 145.98, 145.60, 144.77,144.53, 142.98, 141.59, 140.36, 1311;49, 129.81, 129.36, 125.84, 124.60,121.57, 121.32, 118.34, 114.33, 114.17, 109.99, 102.10, 64.79, 61.46,60.66, 60.17, 59.25, 59.01, 55.40, 48.84, 47.86, 42.13, 39.87, 29.00,28.33, 20.67, 16.01, 9.97.

ESI-MS m/z: Calcd. for C₃₉H₄₀N₄O₁₀S: 756.2. Found (M+1)⁺: 757.3.

Example 15

To a solution of intermediate 15 (12 mg, 0.016 mmol) in acetonitrile(0.66 mL) was added at 23° C. water (0.44 mL, 0.015 M, finalconcentration) and AgNO₃ (81 mg, 0.47 mmol). The reaction mixture wasleft under Argon atmosphere at 23° C. for 23 hours. The reaction wasdiluted with dichloromethane and a saturated solution of sodiumbicarbonate and a saturated solution of sodium chloride was added. Theaqueous phase was extracted with dichloromethane and the combinedorganic layers were dried over sodium sulphate, filtered and the solventwas eliminated under reduced pressure. The crude Et-729 was purified byflash column chromatography (eluent dichloromethane/methanol in gradientfrom 100/0 to 3:1) to afford the final product (8.3 mg, 70%) as a whitesolid.

R_(f): 0.07 (dichloromethane/methanol 95:5)

¹H-RMN (300 MHz, CD₃OD): δ 6.59 (s, 1H), 6.44 (s, 1H), 6.40 (s, 1H),6.13 (s, 1H), 6.02 (s, 1H), 5.20 (d, 1H), 4.73 (s, 1H), 4.58 (d, 2H),4.26 (d, 1H), 4.13 (dd, 1H), 3.80 (broad d, 1H), 3.73 (s, 3H), 3.67 (d,1H), 3.59 (s, 3H), 3.22-3.02 (m, 3H), 2.78 (m, 1H), 2.59 (m, 1H), 2.42(m, 2H), 2.31 (s, 3H), 2.30 (s, 3H), 2.05 (m, 1H), 2.04 (s, 3H).

¹³C-RMN (75 MHz, CD₃OD): δ 173.52, 170.26, 148.13, 147.01, 146.92,146.86, 145.04, 142.67, 141.95, 132.04, 129.28, 125.79, 122.77, 122.42,121.40, 116.29, 115.89, 111.63, 103.54, 90.94, 65.53, 61.80, 60.38,58.23, 57.23, 55.76, 47.35, 43.15, 40.70, 28.85, 27.79, 20.49, 16.07,9.38.

ESI-MS m/z: Calcd. for C₃₈H₄₁N₃O₁₁S: 747.2. Found (M+1)⁺: 748.1

Example 16

To a solution of compound 16 (0.5 g, 0.80 mmol),3-hydroxy-4-metoxy-phenethyl amine (924 mg, 2.8 mmol) in ethyl alcoholwas added at 23° C. silica gel (1 g). The reaction mixture was stirredat 23° C. under Argon atmosphere for 16 hours. After this time thesolvent is eliminated under reduced pressure and the crude of thereaction is purified by flash column chromatography (eluent mixtures ofethyl acetate/methylene chloride in gradient from 1:2 to 100% in ethylacetate, final washes in methylene chloride/methyl alcohol 9:1) toafford Et-770 (564 mg, 91%) as a pale yellow solid.

¹H-NMR (300 MHz, CDCl₃): 6.60 (s, 1H), 6.47 (s, 1H), 6.45 (s, 1H), 6.05(s, 1H), 5.98 (s, 1H), 5.02 (d, 1H), 4.57 (bs, 1H), 4.32 (bs, 1H), 4.28(d, 1H), 4.18 (d, 1H), 4.12 (dd, 1H), 3.78 (s, 3H), 3.62 (s, 3H), 3.50(d, 1H), 3.42 (m, 1H), 3.10 (ddd, 1H), 2.94 (m, 2H), 2.79 (m, 1H), 2.61(m, 1H), 2.47 (m, 1H), 2.35 (m, 1H), 2.32 (s, 3H), 2.27 (s, 3H), 2.20(s, 3H), 2.09 (m, 1H), 2.04 (s, 3H).

ESI-MS m/z: Calcd. for C₄₀H₄₂N₄O₁₀S: 770.7. Found (M+H)⁺: 771.2

Example 17

To a solution of Et-770 (45 mg, 0.058 mmol.) in CH₂Cl₂ (3 mL, 0.03 M)was added at 0° C. under Argon atmosphere m-CPBA (15.1 mg, 0.087 mmol).The reaction was stirred at 0° C. for 30 minutes, then a saturatedaqueous solution of sodium bicarbonate was added, then aqueous phase wasextracted with CH₂Cl₂, the organic layers dried over sodium sulphate andthe solvent was eliminated under reduced pressure. The crude waspurified by flash column chromatography (eluent: ethyl acetate/hexane3:1) to afford compound 17 (45.6 mg, 99%).

R_(f): 0.18 (ethyl acetate/hexane 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.63 (s, 1H); 6.51 (s, 1H); 6.47 (s, 1H);6.19 (s, 1H); 6.05 (s, 1H); 6.00 (s, 1H); 4.66 (d, 1H); 4.61 (d, 1H);4.30-4.28 (m, 1H); 4.19 (s, 1H); 4.07 (s, 1H); 3.82 (s, 1H); 3.73 (d,1H); 3.65 (d, 1H); 3.60 (s, 3H); 3.43 (d, 1H); 3.04-2.95 (m, 2H);2.88-2.81 (m, 1H); 2.72-2.55 (m, 3H); 2.48-2.41 (m, 1H); 2.30 (s, 3H);2.25 (s, 3H); 2.23 (s, 3H); 2.05 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 172.0, 169.2, 148.2, 146.8, 146.3, 145.1,144.8, 142.3, 140.8, 130.8, 129.6, 129.5, 124.5, 122.6, 120.2, 120.0,117.8, 114.6, 111.8, 109.5, 102.4, 70.9, 67.8, 61.8, 61.7, 60.9, 60.6,60.0, 55.3, 54.9, 54.7, 41.9, 40.0, 29.9, 29.1, 25.0, 21.0, 16.2, 10.3

ESI-MS m/z: Calcd. for C₄₀H₄₂N₄O₁₁S: 786.2. Found (M+Na)⁺: 809.3.

Example 18

To a solution of compound 17 (45 mg, 0.057 mmol) in CH₃CN/H₂O (6 mL/2mL, 0.007 M) was added at 23° C. AgNO₃ (287.1 mg, 1.71 mmol). Thereaction mixture was stirred under Argon atmosphere and protected fromthe light for 24 hours. The reaction was diluted with CH₂Cl₂ andquenched with an aqueous saturated solution of sodium bicarbonate and anaqueous saturated solution of brine 1:1. The aqueous phase was extractedwith CH₂Cl₂, the organic layers were dried over sodium sulphate and thesolvent was eliminated under reduced pressure. The crude was purified byflash column chromatography to afford Et-759B (23.2 mg, 52%) as a paleyellow solid and some starting material (18.7 mg, 42%) was recuperated.

R_(f): 0.36 (CH₂Cl₂/MeOH 8:0.5)

¹H-RMN (300 MHz, CDCl₃): δ 6.65 (s, 1H); 6.48 (s, 1H); 6.43 (s, 1H);6.20 (s, 1H); 6.04 (d, 1H); 5.97 (s, 1H); 4.78 (s, 1H); 4.70 (d, 1H);4.55 (d, 1H); 4.36 (d, 1H); 4.07-3.98 (m, 1H); 3.83 (s, 3H); 3.77 (d,1H); 3.69-3.63 (m, 1H); 3.61 (s, 3H); 3.46 (d, 1H); 3.22 (d, 1H);3.06-2.82 (m, 4H); 2.66-2.43 (m, 4H); 2.31 (s, 3H); 2.26 (s, 3H); 2.21(s, 3H); 2.04 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.9, 169.3, 148.0, 146.9, 145.0, 144.7,142.2, 141.0, 130.7, 130.1, 129.6, 124.9, 123.0, 120.9, 120.1, 114.6,113.7, 109.5, 102.2, 82.9, 67.9, 63.1, 61.8, 60.5, 57.7, 57.6, 55.9,55.3, 55.1, 41.7, 40.0, 29.9, 29.2, 24.7, 21.0, 16.1, 14.3, 10.2.

ESI-MS m/z: Calcd. for C₃₉H₄₃N₃O₁₂S: 777.84. Found (M−H₂O+H)⁺: 760.2

Example 19

To a solution of compound 16 (100 mg, 0.16 mmol) in THF/H₂O (4.26mL/1.06 mL, 0.03 M) was added at 23° C. under Argon atmosphere CuCl(79.5 mg, 0.80 mmol). The reaction was stirred at 23° C. under Argonatmosphere and protected from the light for 24 hours. The reaction wasdiluted with CH₂Cl₂, quenched with an aqueous saturated solution ofammonium chloride. The aqueous phase was separated and the organic phasewas washed with an aqueous saturated solution of sodium bicarbonate. Theaqueous phase was extracted with CH₂Cl₂, the organic layers were driedover sodium sulphate and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentCH₂Cl₂/MeOH 60:1) to afford Et-594 (70 mg, 71%) as a yellow solid.

R_(f): 0.44 (CH₂Cl₂/MeOH 60:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.53 (s, 1H); 6.49 (s, 1H); 6.07 (s, 1H);6.05 (s, 1H); 5.98 (s, 1H); 5.94 (s, 1H); 5.71 (s, 2H); 5.18 (d, 1H);5.12 (d, 1H); 4.85 (s, 1H); 4.77 (s, 1H) 4.55-4.36 (m, 3H); 4.17-4.11(m, 4H); 3.77 (s, 3H); 3.75 (s, 3H); 3.58 (d, 1H); 3.47 (s, 4H); 3.19(s, 2H); 3.07 (s, 3H); 2.87-2.54 (m, 6H); 2.31 (s, 3H); 2.30 (s, 3H);2.28 (s, 3H); 2.23 (s, 3H); 2.18-2.05 (m, 2H); 2.15 (s, 3H); 2.11 (s,3H); 2.05 (s, 3H); 1.98 (s, 3H).

ESI-MS m/z: Calcd. for C₃₀H₃₂N₂O₁₀S: 612.1. Found (M−H₂O+H)⁺: 595.5

Example 20

To a solution of Et-770 (1.25 g, 1.62 mmol) in CH₃CN/H₂O (64.8 mL/43.2mL, 0.015 M) was added at 23° C. AgNO₃ (8.27 g, 1.71 mmol). The reactionmixture was stirred under Argon atmosphere and protected from the lightfor 24 hours. The reaction was diluted with CH₂Cl₂ and quenched with anaqueous saturated solution of sodium bicarbonate and an aqueoussaturated solution of brine 1:1. The aqueous phase was extracted withCH₂Cl₂, the organic layers were dried over sodium sulphate and thesolvent was eliminated under reduced pressure. The crude was purified byflash column chromatography (CHCl₃/EtOAc/MeOH in gradient from 49:49:2to 48:40:12) to afford Et-743 (1.09 g, 88%) as a yellow solid and somestarting material (75 mg, 6%) was recuperated.

R_(f): 0.2 (CHCl₃/EtOAc/MeOH 49:49:2)

¹H-RMN (300 MHz, CDCl₃): δ 6.60 (s, 1H), 6.46 (s, 1H), 6.44 (s, 1H),6.02 (d, 1H), 5.94 (broad d, 1H), 5.13 (d, 1H), 4.81 (broad s, 1H), 4.50(broad s, 1H), 4.49 (broad s, 1H), 4.16 (dd, 1H), 4.04 (dd, 1H), 3.79(s, 3H), 3.61 (s, 3H), 3.57 (broad d, 1H), 3.22 (broad, d, 1H), 3.12(ddd, 1H), 2.87 (broad s, 1H), 2.85 (broad s, 1H), 2.80 (m, 1H), 2.60(ddd, 1H), 2.47 (ddd, 1H), 2.38 (broad s, 1H), 2.26 (s, 3H), 2.18 (m,1H), 2.17 (s, 3H), 2.03 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 172.6, 168.3, 147.7, 145.1, 144.4, 143.0,141.3, 140.5, 131.5, 129.2, 126.1, 121.9, 120.9, 118.0, 116.0, 114.0,112.5, 109.8, 101.6, 82.1, 64.7, 61.3, 60.3, 57.8, 56.0, 55.1, 54.9,42.2, 41.4, 39.7, 28.8, 24.0, 20.4, 15.8, 9.6.

ESI-MS m/z: Calcd. for C₃₉H₄₃N₃O₁₁S: 761.3. Found (M−H₂O+H)⁺: 744.4

Example 21

To a solution of Et-743 (25 mg, 0.03 mmol) in methyl alcohol (1.5 mL,0.02 M) was added at 23° C. formic acid (11 μL, 0.3 mmol). The solutionwas stirred at 23° C. for 6 hours, then the solvent was eliminated underreduced pressure and the crude was purified by flash columnchromatography (CHCl₃/EtOAc/MeOH 49:49:2) to afford Et-745 (15.8 mg,64%).

R_(f): 0.17 (CHCl₃/EtOAc/MeOH 49:49:2)

¹H-RMN (300 MHz, CDCl₃): δ 6.61 (s, 1H), 6.49 (s, 1H), 6.42 (s, 1H),6.00 (d, 1H), 5.95 (d, 1H), 5.10 (d, 1H), 4.50 (broad s, 1H), 4.38 (d,1H), 4.09 (dd, 1H), 3.79 (s, 3H), 3.60 (s, 3H), 3.38-3.21 (m, 3H),3.17-2.81 (m, 5H), 2.71 (m, 1H), 2.52 (m, 2H), 2.41 (d, 1H), 2.33 (s,3H), 2.24 (s, 3H), 2.22 (s, 3H), 2.12 (m, 1H), 2.02 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 172.43, 168.19, 147.72, 144.79, 144.60,144.50, 142.64, 141.03, 139.77, 131.61, 128.61, 126.07, 122.09, 120.50,118.51, 115.41, 114.50, 112.63, 109.94, 101.41, 64.26, 64.06, 62.21,60.83, 60.01, 55.00, 42.39, 41.76, 40.78, 39.29, 31.39, 29.50, 28.53,25.45, 22.45, 20.30, 15.62, 13.92, 9.48.

ESI-MS m/z: Calcd. for C₃₉H₄₃N₃O₁₀S: 745.3. Found (M+H)⁺: 746.2

Example 22

To a solution of compound 18 (520.8 mg, 0,84 mmol) in CH₂Cl₂ (17 mL,0.05M) under Argon at room temperature, was added acetic anhydride (0.08mL, 0.88 mmol). The reaction was stirred for 30 min and then quenchedwith an aqueous saturated solution of NaHCO₃. The aqueous layer wasextracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄. Flashchromatography (hexane/EtOAc, 1:2, 2:5, 1:3,) gives pure compound 19(96%).

R_(f): 0.2 (Hexane/ethyl acetate 2:3)

¹H-RMN (300 MHz, CDCl₃): δ 6.56 (s, 1H), 6.04 (dd, 2H), 5.78 (s, 1H),5.52 (bd, 1H), 5.02 (d, 1H), 4.58 (ddd, 1H), 4.53 (bs, 1H), 4.27-4.25(m, 2H), 4.19-4.15 (m, 2H), 3.77 (s, 3H), 3.44-3.43 (m, 2H), 2.92-2.90(m, 2H), 2.36-2.02 (m, 2H), 2.36 (s, 3H), 2.30 (s, 3H), 2.16 (s, 3H),2.02 (s, 3H), 1.88 (s, 3H).

hu 13C-RMN (75 MHz, CDCl₃): δ 170.5, 168.8, 168.4, 148.1, 145.8, 143.1,141.0, 140.3, 130.7, 129.9, 129.0, 120.3, 119.0, 117.9, 113.5, 102.0,61.3, 60.3, 60.2, 59.3, 58.9, 54.7, 54.5, 51.9, 41.8, 41.4, 32.4, 23.7,22.8, 20.4, 16.0, 9.5.

ESI-MS m/z: Calcd. for C₃₃H₃₆N₄O₉S: 664.2. Found (M+H)⁺: 665.2

Example 23

To a solution of compound 19 (100 mg, 0.15 mmol) in acetone (15 n3.,0.01M) was added a solution of Fremy's salt (141 mg, 0.52 mmol) in abuffer solution of KH₂PO₄/Na₂HPO₄ (15 mL, 0.035M). After 24 h at 23° C.the reaction mixture was extracted with CH₂Cl₂ and dried over Na₂SO₄.Chromatography (hexane/erhyl acetate 1:2) gives pure compound 20 (101mg, 99%).

R_(f): 0.38 (Hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.10 (d, 1H); 6.01 (d, 1H); 5,68 (d, 1H);4.98 (d, 1H); 4.54-4.50 (m, 1H); 4.43 (s, 1H); 4.21 (s, 1H); 4.15-4.07(m, 3H); 4.09 (s, 3H); 3.47 (s, 1H); 3.42 (d, 1H); 2.88-2.78 (m, 2H);2.47 (d, 1H); 2.25 (s, 3H); 2.22-2.18 (m, 1H); 2.18 (s, 3H); 2.02 (s,3H); 2.01 (s, 3H); 1.87 (s, 3H).

ESI-MS m/z: Calcd. for C₃₃H₃₄N₄O₁₀S: 678.2. Found (M+H)⁺: 679.1

Example 24

To a solution of compound 20 (100 mg, 0.14 mmol) in THF/H₂O 4:1 (5.6 mg,0.009M) was added CuCl (145 mg, 1.47 mmol). After 24 h at 23° C. thereaction was quenched with an aqueous saturated solution of. NH₄Cl, andwashed with brine and an aqueous saturated solution of NaHCO₃, dilutedand extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄.Chromatography (CH₂Cl₂/MeOH 32:1) gives pure compound ET-637-quinone (60mg, 61%).

R_(f): 0.54 (CH₂Cl₂/MeOH 32:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.07 (d, 1H); 5.97 (d, 1H); 5,72 (d, 1H);5.08 (d, 1H); 4.71 (s, 1H); 4.52-4.33 (m, 3H); 4.08 (s, 3H); 4.08-4.01(m, 3H); 3.53 (d, 1H); 3.24 (d, 1H); 2.83-2.69 (m, 2H); 2.46-2.33 (m,1H); 2.24 (s, 3H); 2.16 (s, 3H); 2.02 (s, 3H); 2.00 (s, 3H); 1.87 (s,3H).

¹³C-RMN (75 MHz, CDCl₃): δ 175.5, 162.6, 162.2, 150.0, 138.9, 134.2,133.7, 130.9, 129.9, 119.9, 111.7, 107.5, 94.9, 75.2, 55.3, 53.6, 50.4,48.6, 46.0, 45.8, 34.1, 33.6, 26.8, 22.6, 15.9, 13.4, 12.7

ESI-MS m/z: Calcd. for C₃₂H₃₅N₃O₁₁S: 669.2. Found (M−H₂O+H)⁺: 652.1

Example 25

To a solution of compound 16 (100 mg, 0.16 mmol) in acetona (16 mL,0.01M) was added a solution of Fremy's salt (151 mg, 0.56 mmol) in abuffer solution of KH₂PO₄/Na₂HPO₄ (16 mL, 0.035M). After 24 h at 23° C.the reaction mixture was extracted with CH₂Cl₂ and dried over Na₂SO₄.Chromatography (hexane/ethyl acetate 1′:1) gives pure compound 21 (79mg, 78%).

R_(f): 0.3 hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.12 (d, 1H); 6.03 (d, 1H); 5.05 (d, 1H);4.48 (s, 1H); 4.28 (d, 1H); 4.24 (s, 1H); 4.20-4.16 (m, 1H); 4.05-4.00(m, 1H); 3.98 (s, 3H); 3.59 (t, 1H); 3.38 (d, 1H); 3.35 (d, 1H);2.84-2.64 (m, 2H); 2.40-2.27 (m, 1H); 3.30 (s, 3H); 2.18 (s, 3H); 2.03(s, 3H); 2.02 (s, 3H).

ESI-MS m/z: Calcd. for C₃₁H₂₉N₃O₁₀S: 635.2. Found (M+H)⁺: 636.1

Example 26

To a solution of compound 21 (79 mg, 0.12 mmol) in THF/H₂O 4:1 (4.4 mL,0.009M) was added CuCl (123 mg, 1.24 mmol). After 24 h at 23° C. thereaction was quenched with an aqueous saturated solution of NH₄Cl, andwashed with brine and an aqueous saturated solution of NaHCO₃, dilutedand extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄.Chromatography (CH₂Cl₂/MeOH 32:1) gives pure compound Et-594-quinone (45mg, 59%).

R_(f): 0.6 (CH₂Cl₂/MeOH 32:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.10-5.97 (m, 4H); 5.12 (d, 1H); 5.05 (d,1H); 4.74-4.63 (m, 2H); 4.43-4.26 (m, 2H); 4.22-4.11 (m, 2H); 4.06 (s,6H); 4.05-3.91 (m, 2H); 3.82-3.71 (m, 2H); 3.55-3.20 (m, 2H); 3.03 (s,4H); 2.75-2.62 (m, 2H); 2.56-2.42 (m, 2H); 2.35-2.23 (m, 2H); 2.29 (s,3H); 2.27 (s, 3H); 2.18 (s, 3H); 2.17 (s, 3H); 2.03 (s, 3H); 2.02 (s,3H); 2.00 (s, 3H); 1.98 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 187.6, 187.2, 186.3, 186.1, 170.0, 169.3,158.5, 158.4, 156.5, 146.6, 146.6, 142.0, 141.9, 140.9, 140.7, 137.4;137.1, 136.7, 129.7, 129.4, 118.6, 117.9, 117.7, 115.1, 114.9, 102.5,102.4, 92.4, 83.0, 82.7, 65.3, 64.6, 60.7, 60.6, 58.8, 58.5, 58.1, 57.5,57.4, 56.0, 55.8, 55.1, 53.7, 53.3, 53.1, 52.6, 42.6, 42.4, 40.9, 40.8,35.5, 35.2, 29.9, 20.9, 20.8, 20.2, 9.9, 9.8

ESI-MS m/z: Calcd. for C₃₀H₃₀N₂O₁₁S: 626.2. Found (M−H₂O+H)⁺: 609.1

Example 27

To a solution of compound 16 (75 mg, 0.12 mmol) in acetic acid (1.5 mL,0.08 M) under Argon at 23° C. was added tryptamine (68 mg, 0.42 mmol).The reaction mixture was stirred at 23° C. for 24 h and then the aceticacid was evaporated. An aqueous saturated solution of NaHCO₃ was addedand the mixture was extracted with CH₂Cl₂. The combined organic layerswere dried over Na₂SO₄. Flash chromatography (hexane/ethyl acetate 1:1)gives pure compound 22 (90 mg, 99%).

R_(f): 0.4 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.74 (s, 1H); 7.38 (d, 1H); 7.25(d, 1H);7.08(t, 1H); 7.00(t, 1H); 6.66(s, 1H); 6.22(d, 1H); 6.02(d, 1H); 5.79(s,1H); 5.08(d, 1H); 4.55(s, 1H); 4.32(s, 1H); 4.27(d, 1H); 4.21(s, 1H);4.19(d, 1H); 3.81(s, 3H); 3.44-3.40(m, 2H); 3.18-2.78(m, 4H);2.71-2.51(m, 3H); 2.37(s, 3H); 2.26(s, 3H); 2.21(s, 3H); 2.06 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.7, 168.9, 148.2, 145.9, 143.2, 141.3,140.5, 135.7, 130.8, 130.6, 129.5, 127.0, 122.2, 120.9, 120.8, 119.5,118.6, 118.4, 113.8, 111.1, 110.5, 102.2, 62.5, 61.5, 60.8, 60.5, 59.7,55.9, 54.8, 42.1, 41.7, 40.0, 39.5, 29.9, 24.0, 21.7, 20.8, 16.1, 9.9.

ESI-MS m/z: Calcd. for C₄₁H₄₁N₅O₈S: 763.3. Found (M+H)⁺: 764.2

Example 28

To a solution of compound 22 (100 mg, 0.13 mmol) in acetone (13 mL,0.01M) was added a solution of Fremy's salt (122 mg, 0.45 mmol) in abuffer solution of KH₂PO₄/Na₂HPO₄ (13 mL, 0.035M). After 24 h at 23° C.the reaction mixture was extracted with CH₂Cl₂ and dried over Na₂SO₄.Chromatography (hexane/ethyl acetate 1:1) gives pure compound 23 (85 mg,85%)

R_(f): 0.4 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.76 (s, 1H); 7.39 (d, 1H); 7.24 (d, 1H);7.09 (ddd, 1H); 7.01 (ddd, 1H); 6.22 (d, 1H); 6.02 (d, 1H); 4.98 (d,1H); 4.44 (s, 1H); 4.22 (s, 1H); 4.19-4.18 (m, 1H) 3.13 (d, 1H); 4.11(s, 3H); 4.04 (d, 1H); 3.48 (s, 1H); 3.39 (d, 1H); 3.16-3.09 (m, 1H);2.88-2.78 (m, 2H); 2.70-2.47 (m, 5H); 2.23 (s, 3H); 2.21 (s, 3H); 2.08(s, 3H); 2.05 (s, 3H).

ESI-MS m/z: Calcd. for C₄₁H₃₉N₅O₉S: 777.3. Found (M+H)⁺: 778.2

Example 29

To a solution of compound 23 (85 mg, 0.10 mmol) in CH₃CN/H₂O 3:2 (5.8mL, 0.009M) was added AgNO₃ (549 mg, 3.27 mmol). After 24 h at 23° C.the reaction was quenched with a mixture 1:1 of an aqueous saturatedsolution of brine and NaHCO₃, stirred for 10 min and diluted andextracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄.Chromatography (CH₂Cl₂/MeOH 32:1) gives pure compound ET-736-quinone (40mg, 50%)

R_(f): 0.6 (CH₂Cl₂/MeOH 32:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.71(s, 1H); 7.39 (d, 1H); 7.26 (d, 1H); 7.10(ddd, 1H); 7.01 (ddd, 1H); 6.22 (d, 1H); 6.01 (d, 1H); 5.12 (d, 1H);4.76 (s, 1H); 4.42-4.37 (m, 2H); 4.11-4.04 (m, 2H) 4.10 (s, 3H); 3.56(s, 1H); 3.35-3.11 (m, 2H); 2.85-2.65 (m, 3H); 2.60-2.36 (m, 4H); 2.23(s, 3H); 2.19 (s, 3H); 2.09 (s, 3H); 2.05 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 186.6, 182.8, 170.9, 169.0, 157.1, 146.1,141.7, 140.9, 137.5, 136.9, 135.7, 130.6, 128.2, 127.0, 122.4, 119.2,119.4, 118.7, 115.3, 111.2, 82.7, 62.9, 62.8, 60.9, 58.6, 57.7, 55.9,53.3, 41.5, 41.1, 40.9, 40.3, 29.9, 22.0, 20.8, 20.0, 14.4, 9.9, 9.1

ESI-MS m/z: Calcd. for C₄₀H₄₀N₄O₁₀S: 768.3. Found (M−H₂O+H)⁺: 751.2

Example 30

To a solution of compound 12 (99.1 mg, 0.15 mmol) in CH₂Cl₂ (3 mL,0.05M) under Argon at 23° C., was added acetic anhydride (0.015 mL, 0.16mmol). The reaction mixture was stirred for 45 min and then quenchedwith an aqueous saturated solution of NaHCO₃. The aqueous layer wasextracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄. Flashchromatography (hexane/EtOAc, in gradient from 3:2 to 1:2) gives purecompound 24 (97 mg, 91%).

R_(f): 0.3 (hexane/ethyl acetate 3:2)

¹H-RMN (300 MHz, CDCl₃): δ 6.54 (s, 1H), 6.02 (d, 2H), 5.96 (s, 1H),5.89-5.76 (m, 1H), 5.52 (d, 1H), 5.11-4.99 (m, 3H), 4.58-4.55 (m, 1H),4.52 (m, 1H), 4.33 (d, 1H), 4.26 (s, 1H), 4.18 (s, 1H), 4.16 (d, 1H),3.74 (s, 3H), 3.54 (bd, 1H), 3.41 (d, 1H), 2.94-2.71 (m, 4H), 2.30-2.25(m, 1H), 2.30 (s, 3H), 2.25 (s, 3H), 2.10 (d, 1H), 2.00 (s, 3H), 1.87(s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 170.5, 168.8, 168.4, 147.8, 145.7, 143.0,140.9, 140.3, 134.8, 131.1, 129.0, 120.3, 120.1, 119.4, 118.1, 117.8,113.4, 113.3, 101.9, 61.2, 60.3, 60.2, 59.4, 59.1, 55.7, 53.0, 51.8,51.7, 41.7, 32.4, 23.8, 22.8, 20.4, 16.0, 9.5.

ESI-MS m/z: Calcd. for C₃₅H₃₈N₄O₉S: 690.2. Found (M+H)⁺: 691.5

Example 31

To a solution of compound 12 (130 mg, 0.20 mmol) in CH₂Cl₂ (3 mL, 0.03M)under Argon atmosphere was added trifluoroacetic anhydride (0.057 mL,0.40 mmol). The reaction mixture was stirred for 30 min at 23° C., thendiluted with CH₂Cl₂, washed with an aqueous saturated solution of NaHCO₃and the organic phase dried over Na₂SO₄. Flash chromatography(hexane/EtOAc, 3:2) gives pure compound 25 (104 mg, 73%).

R_(f): 0.68 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.55 (s, 1H), 6.41 (d, 1H), 6.09 (d, 1H),5.99 (d, 1H); 5.90-5.77 (m, 1H); 5.72 (s, 1H), 5.12-5.03 (m, 3H), 4.60(bp, 1H), 4.54-4.51 (m, 1H), 4.34 (dd, 1H), 4.33 (s, 1H); 4.21 (dd, 1H),4.19 (d, 1H); 3.73 (s, 3H), 3.57-3.55 (m, 1H), 3.44 (d, 1H); 2.87-2.71(m, 4H), 2.43-2.38 (m, 1H), 2.28 (s, 3H), 2.27 (s, 3H), 2.11-2.04 (m,1H); 2.02 (s, 3H).

ESI-MS m/z: Calcd. for C₃₅H₃₅F₃N₄O₉S: 744.2. Found (M+H)⁺: 745.5

Example 32

To a solution of compound 12 (60 mg, 0.09 mmol) in CH₂Cl₂ (3.2 mL,0.03M) under Argon atmosphere were added pyridine (0.008 mL, 0.09 mmol)and palmitoyl chloride (0.03 mL, 0.09 mmol). The reaction mixture wasstirred for 30 min at 23° C., then diluted with CH₂Cl₂ and washed withan aqueous saturated solution of NaHCO₃. The organic phase was driedover Na₂SO₄. Flash chromatography (hexane/EtOAc, 3:2) gives purecompound 26 (71 mg, 90%).

R_(f): 0.62 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.55 (s, 1H), 6.08 (d, 1H), 5.98 (d, 1H),5.89-5.77 (m, 1H); 5.75 (s, 1H); 5.47 (d, 1H), 5.11 (d, 1H); 5.07 (d,1H); 5.02 (d, 1H), 4.62-4.58 (m, 1H), 4.53 (s, 1H), 4.34 (d, 1H), 4.28(s, 1H), 4.19 (d, 1H); 4.17 (dd, 1H); 3.76 (s, 3H), 3.55 (d, 1H), 3.43(d, 1H); 2.96-2.72 (m, 4H), 2.32 (s, 3H), 2.27 (s, 3H), 2.14-1.98 (m,1H); 2.02 (s, 3H), 1.62-1.56 (m, 2H), 1.32-1.28 (m, 24H), 0.87 (t, 3H).

ESI-MS m/z: Calcd. for C₄₉H₆₆N₄O₉S: 886.5. Found (M+H)⁺: 887.9

Example 33

To a solution of compound 12 (65 mg, 0.1 mmol) in CH₂Cl₂ (1.6 mL, 0.06M)under Argon atmosphere were added pyridine (0.009 mL, 0.11 mmol) andpropionyl chloride (0.009 mL, 0.11 mmol). The reaction mixture wasstirred for 15 min at 23° C. then diluted with CH₂Cl₂ and washed with anaqueous saturated solution of NaHCO₃. The organic phase was dried overNa₂SO₄. Flash chromatography (hexane/EtOAc in gradient from 1:1 to 1:2)gives pure compound 27 (41 mg, 59%).

R_(f): 0.66 (hexane/ethyl acetate 1:4)

¹H-RMN (300 MHz, CDCl₃): δ 6.55 (s, 1H), 6.08 (d, 1H), 5.98 (d, 1H);5.90-5.77 (m, 1H); 5.51 (d, 1H), 5.12 (d, 2H), 5.03 (d, 2H), 4.61-4.54(m, 2H), 4.36 (d, 1H), 4.29 (s, 1H), 4.16 (dd, 2H), 3.76 (s, 3H), 3.55(d, 1H), 3.43 (d, 1H), 2.89-2.76 (m, 5H), 2.31 (s, 3H), 2.24 (s, 3H),2.15-2.04 (m, 2H), 2.02 (s, 3H), 1.10 (t, 3H).

ESI-MS m/z: Calcd. for C₃₆H₄₀N₄O₉S: 704.3. Found (M+H)⁺: 705.6

Example 34

To a solution of compound 12 (64 mg, 0.1 mmol) in CH₂Cl₂ (2 mL, 0.05M)under Argon atmosphere were added coumarine acid (23 mg, 0.13 mmol),DIPEA (0.05 mL, 0.15 mmol) and EDC.HCl (60 mg, 0.15 mmol). The reactionmixture was stirred under Argon atmosphere at 23° C. for 4 hours, thendiluted with CH₂Cl₂, washed with an aqueous saturated solution ofNaHCO₃. The organic phase was dried over Na₂SO₄. Flash chromatography(hexane/EtOAc, 1:1) gives pure compound 28 (40 mg, 49%).

R_(f): 0.5 (hexane/ethyl acetate 1:2)

¹H-RMN (300 MHz, CDCl₃): δ 8.98 (d, 1H), 7.69-7.62 (m, 2H), 7.44 (7.33(m, 2H), 6.65 (s, 1H), 6.10 (s, 1H), 5.99 (s, 1H), 5.89-5.80 (m, 1H),5.60 (d, 1H), 5.11-5.05 (m, 4H), 4.69 (dd, 1H), 4.59 (bs, 1H), 4.35-416(m, 5H), 3.67 (3H), 3.58 (d, 1H), 3.45 (d, 2H), 2.93-2.62 (m, 5H), 2.27(s, 3H), 2.13 (s, 3H), 2.03 (s, 3H).

ESI-MS m/z: Calcd. for C₄₃H₄₀N₄O₁₁S: 820.2. Found (M+H)⁺: 821.8

Example 35

To a solution of compound 25 (40 mg, 0.05 mmol) in CH₂Cl₂ (1.5 mL,0.03M) under Argon atmosphere were added Et₃N (0.035 mL, 0.24 mmol) andcinnamoyl chloride (27.7 mg, 0.016 mmol). After 30 min at 23° C., thereaction was diluted with CH₂Cl₂, washed with an aqueous saturatedsolution of NaHCO₃ and the organic phase dried over Na₂SO₄. Flashchromatography (hexane/EtOAc, 3:2) gives pure compound 29 (46 mg, 99%).

R_(f): 0.5 (hexane/ethyl acetate 2:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.93 (d, 1H); 7.62-7.59 (m, 2H); 7.46-7.44(m, 3H); 6.91 (s, 1H), 6.63 (d, 1H); 6.44 (d, 1H), 6.11 (d, 1H), 6.01(d, 1H); 5.84-5.71 (m, 1H); 5.19-5.05 (m, 3H), 4.59-4.53 (m, 1H), 4.35(s, 1H), 4.25-4.21 (m, 2H); 4.00 (d, 1H), 3.71 (s, 3H), 3.64-3.62 (m,1H), 3.49 (d, 1H); 2.95-2.88 (m, 3H), 2.77-2.70 (m, 1H); 2.52-2.48 (m,1H), 2.36 (s, 3H), 2.30 (s, 3H), 2.14-2.04 (m, 1H); 2.06 (s, 3H).

ESI-MS m/z: Calcd. for C₄₄H₄₁F₃N₄O₁₀S: 874.3. Found (M+H)⁺: 875.6

General Experimental Procedure for the Synthesis of Compounds 30, 32,32, 33 and 34: Deallylation Reaction

To a solution of starting material in CH₂Cl₂ (0.04M) were added(PPh₃)₂PdCl₂ (0.08 equiv) and acetic acid (5 equiv). HSnBu₃ (initialamount 8 equiv) was dropwise added at 23° C. and under Argon atmosphereAdditional HSnBu₃ (final amount 48 equiv) was dropwise added during thereaction time (1 hour to 1 hour and 45 min). After this time, thesolution was poured onto a column. Flash chromatography (mixtureshexane/ethyl acetate) gives pure compounds.

Example 36

R_(f): 0.3 (hexane/ethyl acetate 2:5)

¹H-RMN (300 MHz, CDCl₃): δ 6.57 (s, 1H), 6.04 (d, 2H), 5.51 (d, 1H),5.03 (d, 1H), 4.60-4.57 (m, 1H), 4.52 (bp, 1H), 4.47 (d, 1H), 4.27 (s,1H), 4.19-4.15 (m, 2H), 3.84 (bd, 1H), 3.75 (s, 3H), 3.43 (d, 1H), 3.07(s, 1H), 2.96 (q, 1H), 2.34-2.27 (m, 1H), 2.30 (s, 3H), 2.27 (s, 3H),2.13 (d, 1H), 2.02 (s, 3H), 1.87 (s, 3H)

ESI-MS m/z: Calcd. for C₃₂H₃₄N₄O₉S: 650.2. Found (M+H)⁺: 651.4

Example 37

R_(f): 0.3 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.54 (s, 1H), 6.40 (d, 1H), 6.10 (d, 1H),6.00 (d, 1H); 5.05 (d, 1H), 4.60 (bp, 1H), 4.54-4.51 (m, 1H), 4.48 (d,1H), 4.32 (s, 1H); 4.22 (dd, 1H), 4.19 (d, 1H); 3.86-3.84 (m, 1H); 3.73(s, 3H), 3.46 (d, 1H); 2.99-2.98 (m, 2H), 2.44-2.39 (m, 1H), 2.29 (s,3H), 2.26 (s, 3H), 2.13-2.08 (m, 1H); 2.03 (s, 3H).

ESI-MS m/z: Calcd. for C₃₂H₃₁F₃N₄O₉S: 704.2. Found (M+H)⁺: 705.6

Example 38

R_(f): 0.4 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.57 (s, 1H), 6.09 (d, 1H), 5.99 (d, 1H),5.78 (s, 1H); 5.47 (d, 1H), 5.03 (d, 1H); 4.62-4.60 (m, 1H), 4.53 (s,1H), 4.47 (d, 1H), 4.28 (s, 1H), 4.19 (d, 1H); 4.17 (dd, 1H); 3.85 (d,1H); 3.76 (s, 3H), 3.44 (d, 1H); 3.11-2.91 (m, 2H), 2.31 (s, 3H), 2.28(s, 3H), 2.16-1.96 (m, 3H); 2.02 (s, 3H), 1.62-1.56 (m, 2H), 1.38-1.28(m, 24H), 0.87 (t, 3H).

ESI-MS m/z: Calcd. for C₄₆H₆₂N₄O₉S: 846.4. Found (M+H)⁺: 847.0

Example 39

R_(f): 0.2 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.57 (s, 1H), 6.01 (s, 1H), 5.74 (d, 1H),5.52 (d, 1H), 5.10-4.83 (m, 2H), 4.61-4.54 (m, 1H), 4.48 (d, 1H),4.34-4.15 (m, 4H), 3.76 (s, 3H), 3.59-3.34 (m, 3H), 3.10-2.89 (m, 3H),2.30 (s, 3H), 2.29 (s, 3H), 2.12 (dd, 2H), 2.03 (s, 3H), 1.10 (t, 3H).

ESI-MS m/z: Calcd. for C₃₃H₃₆N₄O₉S: 664.2. Found (M+H)⁺: 665.6

Example 40

R_(f): 0.4 (hexane/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.93 (d, 1H); 7.62-7.59 (m, 2H); 7.46-7.44(m, 3H); 6.87 (s, 1H), 6.65 (d, 1H); 6.43 (d, 1H), 6.12 (d, 1H), 6.03(d, 1H); 5.03 (d, 1H); 4.56-4.49 (m, 1H), 4.35 (s, 1H), 4.23-4.1 (m,3H); 3.72 (s, 3H), 3.49 (d, 1H); 3.19-2.90 (m, 3H), 2.52-2.43 (m, 1H),2.30 (s, 3H), 2.147-2.08 (m, 1H); 2.06 (s, 3H), 2.03 (s, 3H).

ESI-MS m/z: Calcd. for C₄₁H₃₇F₃N₄O₁₀S: 834.2. Found (M+H)⁺: 836.0

Example 41

To a solution of compound 30 (25 mg, 0.04 mmol) in CH₂Cl₂ (0.8 mL,0.05M) under Argon atmosphere was added at 23° C. acetic anhydride(0.005 mL, 0.042 mmol). After 1 h at 23° C. more acetic anhydride wasadded (0.005 mL, 0.042 mmol). The reaction was stirred for 4 h more andthen quenched with an aqueousa saturated solution of NaHCO₃. The aqueouslayer was extracted with CH₂Cl₂ and the organic layer was dried overNa₂SO₄. Flash chromatography (hexane/EtOAc, in gradient from 2:5 to1:5,) gives pure compound 35 (24 mg, 90%)

R_(f): 0.15 (hexane/ethyl acetate 2:7)

¹H-RMN (300 MHz, CDCl₃): δ 6.56, 6.54 (2s, 1H), 6.36, 6.15-6.00 (m, 3H),5.52-5.42 (m, 1.6H), 5.29-5.25 (m, 0.6H), 5.07-5.01 (m, 1H), 4.71-4.58(m, 2.4H), 4.30-4.16 (m, 3.4H), 3.76, 3.74 (2s, 3H), 3.41-3.13 (m, 3H),2.40-2.35 (m, 1H), 2.13 (d, 1H), 2.28, 2.27, 2.19, 2.07, 2.02, 1.98,1.87, 1.86 (8s, 15H).

ESI-MS m/z: Calcd. for C₃₄H₃₆N₄O₁₀S: 692.2. Found (M+H)⁺: 693.3

General Experimental Procedure for the Synthesis of Compounds 36, 32,38, 39, 40 and 41: Conversion of the Nitrite Group into Hydroxyl Group.

To a solution of starting material in THF/H₂O 4:1 (0.03M) were added 10equiv. of CuCl. The reaction was stirred for 24 h protected from thelight. After this time, the reaction was quenched with an aqueoussaturated solution of NH₄Cl and diluted with CH₂Cl₂. The organic phaseis washed with brine and an aqueous saturated solution of NaHCO₃ and theaqueous phase extracted with CH₂Cl₂. The combined organic layers weredried over Na₂SO₄. Flash chromatography (mixtures CH₂Cl₂/MeOH) givespure compounds.

Example 42

R_(f): 0.11 (CH₂Cl₂/MeOH 30:1)

¹H-RMN (300 MHz, CD₃OD): δ 6.56 (s, 1H), 6.05 (d, 2H), 5.23 (d, 1H),4.71 (s, 1H), 4.51 (m, 1H), 4.50-4.40 (m, 2H), 4.14-4.11 (m, 2H), 3.71(s, 3H), 3.62-3.60 (m, 1H), 3.52-3.50 (m, 1H), 3.00 (d, 2H), 2.30 (s,3H), 2.28 (s, 3H), 2.28-2.05 (m, 2H), 2.00 (s, 3H), 1.90 (s, 3H).

ESI-MS m/z: Calcd. for C₃₁H₃₅N₃O₁₀S: 641.2. Found (M−H₂O+H)⁺: 624.3

Example 43

R_(f): 0.35 (CH₂Cl₂/MeOH 16:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.55 (s, 1H); 6.49-6.47 (m, 1H); 6.07 (d,1H); 5.97 (d, 1H); 5.18 (d, 1H); 4.83 (s, 1H); 4.52-4.49 (m, 2H); 4.35(sa, 1H); 4.15-4.08 (m, 2H); 3.73 (s, 3H); 3.60-3.45 (m, 2H); 2.96-2.85(m, 2H); 2.47-2.39 (m, 1H); 2.29 (s, 3H); 2.27 (s, 3H); 2.17-2.08 (m,1H); 2.02 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 169.0, 156.4, 156.1, 146.0, 142.9, 141.3,141.1, 131.8, 130.1, 129.0, 121.7, 120.8, 115.1, 114.6, 102.1, 81.1,68.4, 62.2, 60.4, 57.2, 56.1, 53.1, 42.5, 32.1, 32.0, 29.6, 28.1, 22.9,20.7, 14.3, 9.8

ESI-MS m/z: Calcd. for C₃₁H₃₂F₃N₃O₁₀S: 695.2. Found (M−H₂O+H)⁺: 678.4

Example 44

R_(f): 0.4 (CH₂Cl₂/MeOH 16:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.57 (s, 1H); 6.06 (d, 1H); 5.96 (d, 1H);5.53 (d, 1H); 5.15 (d, 1H); 4.78 (s, 1H); 4.59-4.56 (m, 1H); 4.48-4.42(m, 1H); 4.35 (d, 1H); 4.07 (dd, 1H); 3.98 (dd, 1H); 3.76 (s, 3H);3.60-3.57 (m, 1H); 3.52 (d, 1H); 3.02-2.79 (m, 2H); 2.31 (s, 3H); 2.28(s, 3H); 2.17-2.05 (m, 1H); 2.01 (s, 3H); 1.70-1.63 (m, 2H), 1.36-1.24(m, 24H), 0.87 (t, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 172.2, 170.7, 146.1, 145.8, 142.9, 141.2,141.0, 132.1, 129.2, 125.5, 125.0, 121.4, 121.0, 115.4, 112.8, 102.0,81.2, 61.8, 60.6, 57.0, 56.1, 52.0, 51.3, 48.1, 42.4, 36.6, 32.5, 32.1,30.0, 29.9, 29.8, 29.7, 29.6, 28.0, 27.4, 25.6, 22.9, 20.8, 16.2, 14.3,9.8

ESI-MS m/z: Calcd. for C₄₅H₆₃N₃O₁₀S: 837.4. Found (M−H₂O+H)⁺: 820.8

Example 45

R_(f): 0.22 (CH₂Cl₂/MeOH 5:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.58 (s, 1H); 6.07 (s, 1H); 5.97 (s, 1H);5.76 bs, 1H); 5.57 (d, 1H); 5.16 (d, 1H); 4.79 (s, 1H); 4.60-4.35 (m,4H, 4.08 (d, 1H); 3.76 (s, 3H); 3.64-3.50 (m, 2H); 3.00 (d, 1H); 2.84(dd, 1H); 2.30 (s, 3H); 2.28 (s, 3H); 2.35-2.28 (m, 1H) 2.17-2.04 (m,3H); 2.01 (s, 3H); 1.10 (t, 3H).

ESI-MS m/z: Calcd. for C₃₂H₃₇N₃O₁₀S: 655.2. Found (M−H₂O+H)⁺: 638.4

Example 46

R_(f): 0.35 (CH₂Cl₂/MeOH 16:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.94 (d, 1H); 7.62-7.59 (m, 2H); 7.46-7.44(m, 3H); 6.87 (s, 1H), 6.65 (d, 1H); 6.45 (d, 1H), 6.09 (d, 1H), 6.00(d, 1H); 5.15 (d, 1H); 4.83 (s, 1H); 4.52-4.49 (m, 2H); 4.35 (sa, 1H);4.15-4.08 (m, 2H); 3.73 (s, 3H); 3.60-3.45 (m, 2H); 2.96-2.85 (m, 2H);2.47-2.39 (m, 1H); 2.29 (s, 3H); 2.27 (s, 3H); 2.17-2.08 (m, 1H); 2.02(s, 3H).

ESI-MS m/z: Calcd. for C₄₀H₃₈F₃N₃O₁₁S: 825.2. Found (M−H₂O+H)⁺: 809.5

Example 47

R_(f): 0.2 (CH₂Cl₂/MeOH 60:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.58 (s, 1H), 6.02 (d, 2H), 5.83-5.69 (m,1H), 5.69 (s, 1H), 5.59 (d, 1H), 5.17-4.96 (m, 3H), 4.78 (s, 1H),4.57-4.53 (m, 1H), 4.47-4.25 (m, 3H), 4.09 (dd, 1H), 3.77 (s, 3H), 3.47(d, 1H), 3.34-3.31 (m, 1H), 2.92-2.73 (m, 4H), 2.32-2.27 (m, 1H), 2.32(s, 3H), 2.27 (s, 3H), 2.14 (d, 1H), 2.01 (s, 3H), 1.88 (s, 3H).

ESI-MS m/z: Calcd. for C₃₄H₃₉N₃O₁₀S: 681.2. Found (M−H₂O+H)⁺: 664.6

Example 48

To a solution of compound 13 (39 mg, 0.06 mmol) in CH₂Cl₂ (1.5 mL, 0.040M) were added (PPh₃)₂PdCl₂ (3.4 mg, 0.004 mmol), acetic acid (0.02 mL,0.30 mmol) and finally HsnBu₃ (0.05 mL, 0.2 mmol). After 30 min at 23°C. the reaction was poured onto a column. Chromatography (hexane/ethylacete in gradient from 4:1 to 1:2) gives pure compound 42 (31 mg, 86%).

R_(f): 0.38 (hexane/ethyl acete 3:4)

¹H-RMN (300 MHz, CDCl₃): δ 6.58 (s, 1H); 6.51 (s, 1H); 6.11 (d, 1H);6.08 (d, 1H); 6.02 (d, 1H); 5.99 (d, 1H); 5.78 (sa, 2H); 5.10 (d, 1H);5.05 (d, 1H); 4.65 (s, 1H); 4.54 (s, 1H); 4.50 (d, 1H); 4.47 (d, 1H);4.40 (s, 1H); 4.26 (s, 1H); 4.24 (s, 1H); 4.20 (s, 1H); 4.18 (d, 1H);4.01 (dd, 1H); 3.87-3.81 (m, 2H); 3.76 (s, 3H); 3.74 (s, 3H); 3.56 (d,1H); 3.42 (d, 1H); 3.13-2.81 (m, 6H); 2.57 (d, 2H); 2.32 (s, 3H); 3.31(s, 3H); 2.28 (s, 3H); 2.24 (s, 3H); 2.19-2.15 (m, 1H); 2.04 (s, 3H);2.03 (s, 3H).

ESI-MS m/z: Calcd. for C₃₀H₂₉N₃O₉S: 607.2. Found (M+H)⁺: 608.3

Example 49

To a solution of compound 42 (30 mg, 0.05 mmol) in THF/H₂O 4:1 (1.7 mL,0.009M) was added CuCl (49 mg, 0.5 mmol). After 24 h at 23° C. thereaction mixture was quenched with an aqueous saturated solution ofNH₄Cl, diluted with CH₂Cl₂ and washed with brine and an aqueoussaturated solution of NaHCO₃ and the aqueous phase was extracted withCH₂Cl₂. The organic layer was dried over Na₂SO₄. Chromatography(CH₂Cl₂/MeOH 16:1) gives pure compound 43 (3 mg, 10%).

R_(f): 0.2 (CH₂Cl₂/MeOH 16:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.58 (s, 1H); 6.05 (d, 1H); 5.96 (d, 1H);5.78 (sa, 1H); 5.17 (d, 1H); 4.78 (s, 1H); 4.51-4.35 (m, 3H); 4.07 (dd,1H); 4.00 (m, 2H); 3.77 (s, 3H); 3.64-3.53 (m, 2H); 3.07-2.80 (m, 3H);2.31 (s, 3H); 2.17-2.10 (m, 1H); 2.02 (s, 3H).

ESI-MS m/z: Calcd. for C₂₉H₃₀N₂O₁₀S: 598.2. Found 583.1 (M+−H₂O+Me)

Example 50

To a solution of compound 14 (414 mg, 0.5 mmol) in CH₂Cl₂ (9.5 mL,0.032M) under Argon were added Boc₂O (113 mg, 0.5 mmol) and pyridine(0.04 mL, 0.5 mmol). After 2 h at 23° C. more Boc₂O (113 mg, 0.5 mmol)and pyridine (0.04 mL, 0.05 mmol) were added. Additional BOC₂O(113 mg,0.5 mmol) and pyridine (0.04 mL, 0.05 mmol) were added after 3 h. Totalreaction time: 6 hours. The reaction mixture was quenched with anaqueous saturated solution of NaHCO₃, the aqueous phase was extractedwith CH₂Cl₂ and the organic layers dried over Na₂SO₄. Flashchromatography (hexane/ethyl acetate 1:1) gives pure compounds 44 (365mg, 78%) and 45 (105 mg, 20%).

Compound 44

R_(f): 0.5 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.68 (s, 1H); 6.59 (s, 1H); 6.57 (s, 1H);6.03 (d, 1H); 5,96 (d, 1H); 5.93-5.80 (m, 1H); 5.73 (s, 1H); 5.13-5.07(m, 2H); 5.00 (d, 1H); 4.55 (s, 1H); 4.36 (d, 1H); 4.32 (s, 1H); 4.18(d, 1H); 4.09 (dd, 1H); 3.78 (s, 3H); 3.58 (s, 3H); 3.57-3.49 (m, 2H);3.14-3.05 (m, 1H); 2.98-2.76 (m, 4H); 2.68-2.59 (m, 1H); 2.50-2.45 (m,1H); 2.35-2.14 (m, 2H); 2.31 (s, 3H); 2.26 (s, 3H); 2.02 (s, 3H); 1.50(s, 9H).

ESI-MS m/z: Calcd. for C₄₇H₅₂N₄O₁₂S: 896.3. Found (M+H)⁺: 897.0

Compound 45

R_(f): 0.6 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.92 (s, 1H); 6.68 (s, 1H); 6.55 (s, 1H);6.03 (d, 1H); 5,96 (d, 1H); 5.87-5.75 (m, 1H); 5.22-5.07 (m, 2H); 5.00(d, 1H); 4.55 (s, 1H); 4.33 (s, 1H); 4.18 (d, 1H); 4.10 (dd, 1H); 4.06(d 1H); 3.80 (s, 3H); 3.58 (s, 3H); 3.52 (d, 1H); 3.12-3.00 (m, 1H);2.93-2.75 (m, 4H); 2.68-2.58 (m, 1H); 2.51-2.46 (m, 1H); 2.32 (s, 3H);2.31 (s, 3H); 2.27-2.23 (m, 2H); 2.05 (s, 3H); 1.50 (s, 9H); 1,49 (s,9H).

ESI-MS m/z: Calcd. for C₅₂H₆₀N₄O₁₄S: 996.4. Found (M+H)⁺: 997.7

Example 51

To a solution of compound 44 (275 mg, 0.30 mmol) in THF/H₂O 2:1 (15 mL,0.027M) was added an aqueous solution of KOH (4 mL, 1.1 M). The reactionmixture was stirred at 23° C. for 2 h. After this time the reaction wasquenched with brine and extracted with CH₂Cl₂. The organic layer wasdried over Na₂SO₄. Chromatography (hexane/ethyl acete 1:1) gives purecompound 46 (216 mg, 82%).

R_(f): 0.48 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.70 (s, 1H); 6.59 (s, 1H); 6.55 (s, 1H);6.25 (s, 1H); 5.95 (d, 1H); 5.89 (d, 1H); 5.87-5.77 (m, 1H); 5.72 (s,1H); 5.10-5.03 (m, 2H); 4.99 (d, 1H); 4.49 (d, 1H); 4.38-4.36 (m, 2H);4.17 (d, 1H); 4.05 (dd, 1H); 3.68 (s, 3H); 3.59-3.53 (m, 2H); 3.56 (s,3H); 3.13-3.04 (m, 1H); 2.99-2.71 (m, 4H); 2.68-2.46 (m, 3H); 2.40 (d,1H); 2.30 (s, 3H); 2.16 (s, 3H); 1.50 (s, 9H).

ESI-MS m/z: Calcd. for C₄₅H₅₀N₄O₁₁S: 854.3. Found (M+H)⁺: 855.6

Example 52

To a solution of compound 46 (108 mg, 0.13 mmol) in CH₂Cl₂ (4 mL,0.032M) under Argon atmosphere at 23° C. were added pyridine (0.02 mL,0.26 mmol) and the cinnamoyl chloride (21 mg, 0.13 mmol). The reactionmixture was left for 2 hours at 23° C. and quenched after this time withan aqueous saturated solution of NaHCO₃, the aqueous phase extractedwith CH₂Cl₂ and the organic layers dried over Na₂SO₄. Flashchromatography (hexane/ethyl acetate 2:1) gives pure compound 47 (53 mg,43%)

R_(f): 0.67 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.88 (d, 1H); 7.59-7.57 (m, 2H); 7.44-7.39(m, 3H); 6.71 (s, 1H); 6.589 (d, 1H); 6.58 (s, 1H); 6.54 (s, 1H); 6.06(d, 1H); 5.97 (d, 1H); 5.92-5.79 (m, 1H); 5.46 (s, 1H); 5.13-5.05 (m,2H); 5.01 (d, 1H); 4.57 (s, 1H); 4.37-4.34 (m, 2H); 4.20 (s, 1H); 4.11(d, 1H); 3.61 (s, 3H); 3.55 (d, 2H); 3.45 (s, 3H); 3.15-3.09 (m, 1H);2.96-2.62 (m, 4H); 2.51-2.31 (m, 3H); 2.25 (s, 3H); 2.09 (s, 3H); 1.51(s, 9H)

ESI-MS m/z: Calcd. for C₅₅H₅₆N₄O₁₂S: 984.4. Found (M+H)⁺: 986.0

Example 53

To a solution of compound 46 (108 mg, 0.13 mmol) in CH₂Cl₂ (4 mL,0.032M) under Argon atmosphere were added octanoic acid (0.02 mL, 0.13mmol), DMAP (31 mg, 0.26 mmol) and EDC.HCl (48 mg, 0.26 mmol). Thereaction was stirred at 23° C. for 2 h. After this time the reactionmixture was diluted with CH₂Cl₂, washed with brine and the organic layerdried over Na₂SO₄. Flash chromatography (hexane/ethyl acetate 2: 1)gives pure compound 48 (86 mg, 69%).

R_(f): 0.85 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.68 (s, 1H); 6.58 (s, 1H); 6.56 (s, 1H);6.03 (d, 1H); 5.95 (d, 1H); 5.93-5.79 (m, 1H); 5.65 (s, 1H); 5.13-5.07(m, 2H); 5.00 (d, 1H); 4.53 (d, 1H); 4.36-4.32 (m, 2H); 4.17 (d, 1H);4.09 (dd, 1H); 3.76 (s, 3H); 3.58 (s, 3H); 3.57-3.50 (m, 2H); 3.14-3.06(m, 1H); 2.97-2.75 (m, 4H); 2.68-2.45 (m, 3H); 2.35-2.14 (m, 2H); 2.31(s, 3H); 2.01 (s, 3H); 1.75-1.71 (m, 2H); 1.50 (s, 9H); 1.36-1.24 (m,10H); 0.89 (t, 3H).

ESI-MS m/z: Calcd. for C₅₃H₆₄N₄O₁₂S: 980.4. Found (M+H)⁺: 982.0

Example 54

A solution of compound 47 (38 mg, 0.03 mmol) in CH₂Cl₂/H₂O/TFA 2:1:3.3(3.1 mL, 0.013M) was stirred at 23° C. for 64 h. The reaction mixturewas neutralised with an aqueous saturated solution of NaHCO₃, extractedwith CH₂Cl₂ and the organic layers dried over Na₂SO₄. Flashchromatography (hexane/ethyl acetate 3:2) gives pure compound 49 (34 mg,99%).

R_(f): 0.56 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.86 (d, 1H); 7.58-7.54 (m, 2H); 7.46-7.44(m, 3H); 6.57 (d, 1H); 6.55 (s, 1H); 6.49 (s, 1H); 6.45 (s, 1H); 6.07(d, 1H); 5.99 (d, 1H); 5.90-5.79 (m, 1H); 5.42 (s, 1H); 5.13-5.04 (m,2H); 5.03 (d, 1H); 4.60 (s, 1H); 4.37-4.34 (m, 2H); 4.23-4.20 (m, 2H);4.13 (d, 1H); 3.64 (s, 3H); 3.55 (d, 2H); 3.44 (s, 3H); 3.15-3.06 (m,1H); 2.97-2.77 (m, 4H); 2.64-2.34 (m, 4H); 2.24 (s, 3H); 2.09 (s, 3H).

ESI-MS m/z: Calcd. for C₄₉H₄₈N₄O₁₀S: 884.3. Found (M+H)⁺: 885.0

Example 55

A solution of compound 48 (65 mg, 0.06 mmol) in CH₂Cl₂/H₂O/TFA 2:1:3.3(5.3 mL, 0.013M) was stirred at 23° C. for 64 h. The reaction mixturewas neutralised with an aqueous saturated solution of NaHCO₃, extractedwith CH₂Cl₂ and the organic layer dried over Na₂SO₄. Flashchromatography (hexane/ethyl acetate 3:2) gives pure compound 50 (57 mg,99%).

R_(f): 0.64 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.59 (s, 1H); 6.47 (s, 1H); 6.43 (s, 1H);6.04 (d, 1H); 5.96 (d, 1H); 5.91-5.80 (m, 1H); 5.64 (s, 1H); 5.14-5.07(m, 2H); 5.00 (d, 1H); 4.55 (d, 1H); 4.36-4.33 (m, 2H); 4.20 (d, 1H);4.11 (dd, 1H); 3.77 (s, 3H); 3.62 (s, 3H); 3.58-3.50 (m, 2H); 3.12-3.07(m, 1H); 2.98-2.76 (m, 4H); 2.63-2.43 (m, 3H); 2.36-2.10 (m, 2H); 2.31(s, 3H); 2.02 (s, 3H); 1.73-1.65 (m, 2H); 1.34-1.20 (m, 10H); 0.89 (t,3H).

ESI-MS m/z: Calcd. for C₄₈H₅₆N₄O₁₀S: 880.4. Found (M+H)⁺: 882.0

Synthesis of Compounds 51 and 52 Following the General ExperimentalProcedure for Deallylation Reactions

Example 56

R_(f): 0.26 hexane/ethyl acetate 1:1)

¹H NMR (300 MHz, CDCl₃): 7.87 (d, 1H); 7.59-7.54 (m, 4H); 7.51-7.44 (m,6H); 6.60-6.43 (m, 8H); 6.07 (d, 2H); 5.97 (d, 2H); 5.03 (d, 2H); 4.59(s, 2H); 4.50 (d, 1H); 4.37-4.34 (m, 2H); 4.23-4.09 (m, 4H); 3.84 (d,2H); 3.65 (s, 3H); 3.64 (s, 3H); 3.61 (s, 6H); 3.57-3.52 (m, 2H);3.43-3.40 (m, 2H); 3.14-2.97 (m, 6H); 2.93-2.80 (m, 6H); 2.68-2.58 (m,2H); 2.48-2.20 (m, 4H); 2.29 (s, 3H); 2.23 (s, 3H); 2.09 (s, 3H); 1.94(s, 3H).

ESI-MS m/z: Calcd. for C₄₆H₄₄N₄O₁₀S: 844.3. Found (M+H)⁺: 845.0

Example 57

R_(f): 0.36 hexane/ethyl acete 1:1)

¹H-NMR (300 MHz, CDCl₃) δ: 6.61 (s, 1H); 6.47 (s, 1H); 6.43 (s, 1H);6,05 (d, 1H); 5.97 (d, 1H); 5.72 (s, 1H); 5.02 (d, 1H); 4.55 (d, 1H);4.50 (d, 1H); 4.33 (s, 1H); 4.19 (d, 1H); 4.11 (dd, 1H); 3.85 (d, 1H);3.76 (s, 3H); 3.61 (s, 3H); 3.52 (d, 1H); 3.14-2.97 (m, 3H); 2.81-2.76(m, 1H); 2.67-2.48 (m, 3H); 2.43-2.33 (m, 1H); 2.30 (s, 3H); 2.17-2.09(m, 1H); 2.02 (s, 3H); 1.74-1.70 (m, 2H); 1.38-1.20 (m, 10H); 0.89 (t,3H)

ESI-MS m/z: Calcd. for: C₄₅H₅₂N₄O₁₀S.840.3 Found (M+H)⁺: 841.1

General Experimental Procedure for the Synthesis of Compounds 53 and 54.Interconversion of the Nitrile Group into the Hydroxyl Group.

To a solution of starting material in CH₃CN/H₂O 3:2 (0.015M) was addedAgNO₃ (30 equiv). After 24 h at 23° C., the reaction was quenched with amixture 1:1 of saturated aqueous solutions of brine and NaHCO₃, stirredfor 10 min, diluted and extracted with CH₂Cl₂. The organic layer wasdried over Na₂SO₄. Chromatography (mixtures of CH₂Cl₂:MeOH) gives purecompounds 53 and 54.

Example 58

R_(f): 0.46 (CH₂Cl₂/MeOH 8:0.5)

¹H-NMR (300 MHz, CDCl₃): δ 7.87 (d, 1H); 7.59-7.54 (m, 4H); 7.51-7.44(m, 6H); 6.60-6.43 (m, 8H); 6.05 (d, 2H); 5.97 (d, 2H); 5.14 (d, 2H);4.87 (s, 2H); 4.54-4.37 (m, 4H); 4.06-4.02 (m, 2H); 3.74-3.60 (m, 4H);3.64 (s, 3H); 3.63 (s, 3H); 3.61 (s, 3H); 3.48 (s, 3H); 3.48-3.43 (m,2H); 3.18-3.01 (m, 6H); 2.91-2.83 (m, 6H); 2.70-2.58 (m, 2H); 2.49-2.22(m, 4H); 2.29 (s, 3H); 2.24 (s, 3H); 2.08 (s, 3H); 1.93 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 145.7, 144.6, 144.5, 131.1, 129.4, 129.0,128.9, 128.5, 127.1, 126.7, 125.0, 121.6, 117.3, 114.9, 114.2, 110.0,101.9, 81.5, 68.4, 61.4, 60.6, 57.3, 56.2, 55.3, 51.4, 48.0, 42.4, 40.0,38.9, 37.3, 36.0, 33.0, 32.1, 32.0, 31.2, 30.5, 29.6, 29.1, 27.6, 27.3,23.9, 23.2, 22.9, 20.0, 16.0, 14.4, 11.2, 9.8

ESI-MS m/z: Calcd. for: C₄₅H₄₅N₃O₁₁S: 835.3. Found (M+H)⁺: 836.0

Example 59

R_(f): 0.31 (CH₂Cl₂/MeOH 8:0.5)

¹H-NMR (300 MHz, CDCl₃) δ: 6.60 (s, 1H); 6.45 (s, 1H); 6.42 (s, 1H);6.02 (d, 1H); 5.95 (d, 1H); 5.12 (d, 1H); 4.83 (s, 1H); 4.52 (d, 1H);4.45 (s, 1H); 4.40-4.36 (m, 1H); 4.06-3.99 (m, 2H); 3.76 (s, 1H);3.63-3.60 (m, 2H); 3.61 (s, 3H); 3.46 (d, 1H); 3.15-3.00 (m, 3H);2.90-2.77 (m, 2H); 2.63-2.49 (m, 3H); 2.43-2.35 (m, 1H); 2.30 (s, 3H);2.18-2.06 (m, 1H); 2.00 (s, 3H); 1.74-1.70 (m, 2H); 1.34-1.20 (m, 10H);0.89 (t, 3H).

ESI-MS m/z: Calcd. for: C₄₄H₅₃N₃O₁₁S: 831.3 Found (M+H)⁺: 832.0

General Experimental Procedure for Introduction of the TryptamineMoiety, Pictect-Spengler Reaction; Synthesis of Compounds 55, 56, 57 and58.

To a solution of compound 13 in acetic acid (0.5 10⁻⁴M) under Argonatmosphere at 23° C. was added the tryptamine reagent. The reactionmixture was stirred for 24 h at 23° C. (for compounds 57 and 58temperature reaction 60° C.) and then the acetic acid was evaporated. Anaqueous saturated solution of NaHCO₃ was added and the mixture wasextracted with CH₂Cl₂ and the organic layers were dried over Na₂SO₄.Flash chromatography (mixtures of hexane/ethyl acetate) gives purecompounds.

Example 60

R_(f): 0.45 (hexane/ethyl acete 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.74 (s, 1H); 7.38 (d, 1H); 7.24 (d, 1H);7.09 (t, 1H); 7.00 (t, 1H); 6.66 (s, 1H); 6.22 (d, 1H); 6.01 (d, 1H);5.94-5.80 (m, 1H); 5.78 (s, 1H); 5.15-5.07 (m, 3H); 4.56 (s, 1H); 4.37(d, 1H); 4.33 (s, 1H); 4.23(d, 1H); 4.19 (dd, 1H); 3.80 (s, 3H); 3.55(d, 1H); 3.44 (d, 1H); 3.17-2.80 (m, 6H); 2.71-2.52 (m, 3H); 2.37 (s,3H); 2.25 (s, 3H); 2.06 (s, 3H).

ESI-MS m/z: Calcd. for C₄₃H₄₃N₅O₈S: 789.3. Found (M+H)⁺: 790.0

Example 61

R_(f): 0.18 (Hex/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.65 (s, 1H); 7.11 (d, 1H); 6.81 (d, 1H);6.73 (dd, 1H); 6.66 (s, 1H); 6.19 (d, 1H); 5.99 (d, 1H); 5.92-5.83 (m,1H); 5.78 (s, 1H); 5.15-5.06 (m, 3H); 4.55 (s, 1H); 4.36 (d, 1H); 4.32(s, 1H); 4.22(d, 1H); 4.18 (dd, 1H); 3.81 (s, 3H); 3.79 (s, 3H); 3.54(d, 1H); 3.44 (d, 1H); 3.16-3.03 (m, 2H); 2.96-2.78 (m, 4H); 2.65-2.50(m, 3H); 2.38 (s, 3H); 2.25 (s, 3H); 2.05 (s, 3H).

ESI-MS m/z: Calcd. for C₄₄H₄₅N₅O₉S: 819.3. Found (M+H)⁺: 820.5

Example 62

R_(f): 0.10 (Hex/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.72 (s, 1H); 6.88 (d, 1H); 6.65 (d, 1H);6.60 (d, 1H); 6.52 (dd, 1H); 6.10 (s, 1H); 5.94 (s, 1H); 5.94-5.81 (m,1H); 5.82 (s, 1H); 5.14-5.03 (m, 3H); 4.53 (s, 1H); 4.35 (d, 1H); 4.29(s, 1H); 4.20(d, 1H); 4.17 (dd, 1H); 3.80 (s, 3H); 3.53 (d, 1H); 3.41(d, 1H); 3.09-3.01 (m, 2H); 2.91-2.72 (m, 5H); 2.56-2.51 (m, 2H); 2.37(s, 3H); 2.23 (s, 3H); 2.03 (s, 3H).

ESI-MS m/z: Calcd. for C₄₃H₄₃N₅O₉S: 805.3. Found (M+H)⁺: 806.5

Example 63

R_(f): 0.48 (Hex/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.66 (s, 1H); 7.14 (S, 1H); 7.13 (d, 1H);6.91 (d, 1H); 6.66 (S, 1H); 6.21 (s, 1H); 6.00 (s, 1H); 5.92-5.81 (m,1H); 5.80 (s, 1H); 5.15-5.06 (m, 3H); 4.55 (s, 1H); 4.37 (d, 1H); 4.32(s, 1H); 4.21(d, 1H); 4.16 (d, 1H); 3.81 (s, 3H); 3.54 (d, 1H); 3.43 (d,1H); 3.17-3.03 (m, 2H); 2.96-2.77 (m, 5H); 2.68-2.42 (m, 4H); 2.37 (s,3H); 2.25 (s, 3H); 2.06 (s, 3H).

ESI-MS m/z: Calcd. for C₄₄H₄₅N₅O₈S: 803.3. Found (M+H)⁺: 804.4

Synthesis of Compounds 59, 60, 61 and 62 Following the GeneralExperimental Procedure for Deallylation Reactions

Example 64

R_(f): 0.21 (Hex/ethyl acetate 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.74 (s, 1H); 7.38 (d, 1H); 7.24 (d, 1H);7.09 (t, 1H); 7.00 (t, 1H); 6.68 (s, 1H); 6.23 (d, 1H); 6.02 (d, 1H);5.10 (d, 1H); 4.55 (s, 1H); 4.51 (d, 1H); 4.33 (s, 1H); 4.23 (d, 1H);4.19 (dd, 1H); 3.84 (d, 1H); 3.80 (s, 3H); 3.45 (d, 1H); 3.25-2.79 (m,6H); 2.71-2.53 (m, 3H); 2.36 (s, 3H); 2.26 (s, 3H); 2.07 (s, 3H).

ESI-MS m/z: Calcd. for C₄₀H₃₉N₅O₈S: 749.3. Found (M+H)⁺: 749.9

Example 65

R_(f): 0.15 (Hex/ethyl acetate 1:2)

¹H-RMN (300 MHz, CDCl₃): δ 7.65 (s, 1H); 7.12 (d, 1H); 6.81 (d, 1H);6.73 (dd, 1H); 6.67 (s, 1H); 6.20 (s, 1H); 6.00 (s, 1H); 5.08 (d, 1H);4.55 (s, 1H); 4.49 (d, 1H); 4.31 (s, 1H); 4.31-4.16 (m, 2H); 3.83 (d,1H); 3.80 (s, 3H); 3.79 (s, 3H); 3.44 (d, 1H); 3.24-3.11 (m, 2H);3.03-2.94 (m, 1H); 2.83-2.80 (m, 1H); 2.65-2.50 (m, 4H); 2.36 (s, 3H);2.26 (s, 3H); 2.06 (s, 3H).

ESI-MS m/z: Calcd. for C₄₁H₄₁N₅O₉S: 779.3. Found (M+H)⁺: 780.0

Example 66

R_(f): 0.10 (Hex/ethyl acetate 1:2)

¹H-RMN (300 MHz, CDCl₃): δ 7.67 (s, 1H); 6.93 (d, 1H); 6.66 (s, 2H);6.55 (dd, 1H); 6.15 (s, 1H); 5.98 (s, 1H); 5.06 (d, 1H); 4.53 (s, 1H);4.48 (d, 1H); 4.29 (s, 1H); 4.19 (d, 1H); 3.82 (d, 1H); 3.78 (s, 3H);3.42 (d, 1H); 3.22-2.96 (m, 5H); 2.76-2.73 (m, 2H); 2.57-2.43 (m, 3H);2.35 (s, 3H); 2.25 (s, 3H); 2.04 (s, 3H).

ESI-MS m/z: Calcd. for C₄₀H₃₉N₅O₉S: 765.3. Found (M+H)⁺: 766.4

Example 67

R_(f): 0.35 (ex/ethyl acetate 1:2)

¹H-RMN (300 MHz, CDCl₃): δ 7.63 (s, 1H); 7.26 (s, 1H); 7.15 (d, 1H);6.91 (d, 1H); 6.68 (s, 1H); 6.22 (d, 1H); 6.02 (d, 1H, 5.09 (d, 1H);4.55 (s, 1H); 4.50 (d, 1H); 4.32 (s, 1H); 4.22 (d, 1H); 4.18 (dd, 1H);3.83 (d, 1H); 3.80 (s, 3H); 3.44 (d, 1H); 3.25-3.10 (m, 3H); 3.03-2.94(m, 1H); 2.83-2.77 (m, 1H); 2.66-2.51 (m, 4H); 2.37 (s, 3H); 2.36 (s,3H); 2.26 (s, 3H); 2.07 (s, 3H).

ESI-MS m/z: Calcd. for C₄₁H₄₁N₅O₈S: 763.3. Found (M+. H)⁺: 764.0

General Experimental Procedure for the Synthesis of Compounds 63, 64,65, 66 and 67. Interconversion of the Nitrile Group into the HydroxylGroup.

To a solution of starting material in CH₃CN/H₂O 3:2 (0.015M) was addedAgNO₃ (30 equiv). After 24 h at 23° C., the reaction was quenched with amixture 1:1 of saturated aqueous solutions of brine and NaHCO₃, stirredfor 10 min. diluted and extracted with CH₂Cl₂. The organic layer wasdried over Na₂SO₄. Chromatography (mixtures of CH₂Cl₂:MeOH) gave purecompounds 63, 64, 65, 66 and 67.

Example 68

R_(f): 0.13 (CH₂Cl₂/MeOH 16:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.72 (s, 1H); 7.38 (d, 1H); 7.24 (d, 1H);7.09 (t, 1H); 7.00 (t, 1H); 6.68 (s, 1H); 6.21 (d, 1H); 6.00 (d, 1H);5.21 (d, 1H); 4.85 (s, 1H); 4.53-4.38 (m, 3H); 4.13-4.08 (m, 2H); 3.80(s, 3H); 3.64-3.57 (m, 3H); 3.17-3.08 (m, 2H); 2.91-2.82 (m, 2H);2.69-2.54 (m, 3H); 2.36 (s, 3H); 2.25 (s, 3H); 2.08 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.7, 146.0, 145.7, 142.8, 141.4, 140.9,135.7, 132.0, 131.1, 129.5, 127.1, 125.0, 124.6, 122.1, 121.8, 121.5,119.4, 118.6, 115.7, 111.2, 110.4, 102.0, 81.7, 62.6, 62.1, 60.6, 57.2,56.1, 51.4, 48.1, 42.6, 40.0, 39.4, 29.9, 27.4, 21.8, 20.8, 16.0, 9.9

ESI-MS m/z: Calcd. for C₃₉H₄₀N₄O₉S: 740.3. Found (M−H₂O+H)⁺: 723.0

Example 69

R_(f): 0.36 (CH₂Cl₂/MeOH 8:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.60 (s, 1H); 7.12 (d, 1H); 6.81 (d, 1H);6.74 (dd, 1H); 6.68 (s, 1H); 6.19 (s, 1H); 6.00 (dd, 1H); 5.20 (d, 1H);4.84 (s, 1H); 4.53-4.37 (m, 3H); 4.12-4.07 (m, 2H); 3.80 (s, 3H); 3.78(s, 3H); 3.60-3.53 (m, 3H); 3.18-3.11 (m, 2H); 2.90-2.79 (m, 2H);2.66-2.49 (m, 3H); 2.36 (s, 3H); 2.25 (s, 3H); 2.05 (s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.7; 168.9; 154.0; 146.1; 145.7; 142.9;141.4; 140.9; 134.0; 131.9; 130.9; 129.6; 129.3; 127.4; 124.5; 121.8;121.5; 115.7; 113.1; 111.9; 110.1; 102.1; 100.6; 81.7; 62.6; 62.0; 60.6;57.1; 56.1; 51.4; 48.0; 42.6; 40.1; 39.4; 29.9; 27.4; 21.8; 20.8; 16.0;9.9.

ESI-MS m/z: Calcd. for C₄₀H₄₂N₄O₁₀S: 770.3. Found (M−H₂O+H)⁺: 753.2

Example 70

R_(f): 0.15 (CH₂Cl₂/MeOH 8:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.59 (s, 1H); 7.03 (d, 1H); 6.72 (d, 1H);6.68 (t, 1H); 7.00 (t, 1H); 6.68 (s, 1H); 6.61 (dd, 1H); 6.18 (s, 1H);5.97 (s, 1H); 5.20 (d, 1H); 4.84 (s, 1H); 4.52-4.36 (m, 3H);4.12-4.09(m, 2H); 3.80 (s, 3H); 3.60-3.48 (m, 3H); 3.16-3.10 (m, 3H);2.91-2.77 (m, 2H); 2.57-2.43 (m, 3H); 2.36 (s, 3H); 2.26 (s, 3H);2.05(s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.7; 169.2; 149.6; 146.1; 142.8; 141.3;141.0; 132.1; 132.0; 131.1; 131.0; 129.6; 129.0; 127.7; 124.6; 121.8;121.5; 115.7; 113.1; 111.7; 109.8; 103.4; 102.1; 81.7; 68.3; 62.0; 60.6;57.1; 56.1; 51.4; 48.0; 42.5; 40.0; 39.2; 32.1; 27.4; 20.8; 16.0; 9.9.

ESI-MS m/z: Calcd. for C₃₉H₄₀N₄O₁₀S: 756.3. Found (M−H₂O+H)⁺: 739.0

Example 71

R_(f): 0.47 (CH₂Cl₂/MeOH 8:1)

¹H-RMN (300 MHz, CDCl₃): δ 7.61 (s, 1H); 7.14 (s, 1H); 7.13 (d, 1H);6.91 (d, 1H); 6.66 (s, 1H); 6.20 (s, 1H); 6.01 (d, 1H); 5.19 (d, 1H);4.85 (s, 1H); 4.54-4.40 (m, 3H); 4.12-4.08 (m, 2H); 3.80 (s, 3H); 3.62(d, 2H); 3.54 (m, 1H); 3.17-3.08 (m, 2H); 2.90-2.78 (m, 2H); 2.64-2.47(m, 3H); 2.37 (s, 3H); 2.36 (s, 3H); 2.25 (s, 3H); 2.06(s, 3H).

¹³C-RMN (75 MHz, CDCl₃): δ 171.7; 168.9; 146.2; 145.7; 142.9; 141.4;140.9; 134.1; 132.4; 132.3; 131.2; 129.6; 128.6; 127.3; 123.7; 121.7;121.4; 118.4; 115.7; 113.1; 110.8; 109.9; 102.1; 81.6; 62.0; 60.6; 56.1;51.4; 48.0; 42.5; 39.4; 32.1; 29.2; 28.1; 27.3; 21.8; 20.8; 16.0; 13.8;9.9.

ESI-MS m/z: Calcd. for C₄₀H₄₂N₄O₉S: 754.3. Found (M−H₂O+H)⁺: 737.3

Example 72

R_(f): 0.26 (CH₂Cl₂/MeOH 30:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.62 (s, 1H), 6.47 (s, 1H), 6.45 (s, 1H),6.03 (s, 1H), 5.95 (s, 1H), 5.78 (m, 1H), 5.67 (s, 1H), 5.38 (m, 1H),5.14 (d, 2H), 5.05 (bs, 1H), 4.99 (bs, 1H), 4.83 (bs, 1H), 4.49 (bs,1H), 4.28 (bs, 1H), 4.05 (d, 1H), 3.79 (s, 3H), 3.61 (s, 3H), 3.57 (m,1H), 3.35 (m, 1H), 3.12 (m, 1H), 2.85 (m, 4H), 2.60 (m, 2H), 2.51 (m,1H), 2.32 (s, 3H), 2.26 (s, 3H), 2.18 (m, 4H), 2.03 (s, 3H)

ESI-MS m/z: Calcd. for C₄₁H₄₅N₃O₁₁S: 787.8. Found (M−H₂O+H)⁺: 770.4

Example 73

A solution of Et-729 (19.9 mg, 0.03 mmol) in a methanolic solution ofKOH (5.21 ml, 0.95 mmol, 0.1817M) was stirred under argon at 23° C.After 1 h the reaction was diluted with CH₂Cl₂ and extracted. Theorganic layer was dried over Na₂SO₄. Chromatography gives pure compound.

R_(f): 0.18 (CH₂Cl₂/MeOH 10:1)

¹H-RMN (300 MHz, CD₃OD): δ 6.61 (s, 1H); 6.39 (s,1H); 6.33 (s, 1H); 6.03(s, 1H); 5.88 (s, 1H); 5-45-5.44 (m, 1H); 5.13 (d, 1H); 4.81 (s, 1H);4.71-6.67 (m, 2H); 4.30 (d, 1H); 4.08 (dd, 1H); 3.92 (d, 1H); 3.79 (d,1H); 3.73 (s, 3H), 3.55 (s, 3H); 3.18-3.05 (m, 3H); 2.83-2.79 (m, 1H);2-70-2.56 (m, 1H); 2.38 (d, 1H); 2.30 (s, 3H); 2.20 (d, 1H); 2.14 (s,3H).

ESI-MS m/z: Calcd. for C₃₆H₃₉N₃O₁₀S: 705.2. Found (M−H₂O+H)⁺: 688.4

Example 74

To a solution of intermediate 1 (1.17 g, 2.26 mmol) and cysteinederivative (0.9 g, 2.26 mmol) in anhydrous dichloromethane (45 mL,0.05M) was added at 23° C. under Argon atmosphere EDC.HCl (0.87 g, 4.52mmol) and DMAP (0.55 g, 4.52 mmol). The reaction mixture was left at 23°C. under Argon atmosphere for 1 hour. A saturated aqueous solution ofsodium bicarbonate was added and the aqueous phase was extracted withdichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentmixtures of dichloromethane/methanol in gradient from 100:0 to 80: 1) toafford intermediate 69 (1.43 g, 70%) as a yellow solid.

R_(f): 0.5 (dichloromethane/MeOH 60:1)

¹H-RMN (CDCl₃, 300 MHz) δ 7.63 (d, 2H), 7.47 (dd, 2H), 7.34 (m, 2H),7.23 (m, 2H), 6.35 (s, 1H), 6.09 (d, 1H), 5.91 (m, 2H), 5.85 (d, 1H),5.62 (m, 1H), 5.58 (s, 1H), 5.30 (dd, 1H), 5.20 (dd, 1H), 4.15 (d, 1H),4.06-3.91 (m, 4H), 3.78 (dd, 1H), 3.69 (broad t, 1H), 3.49 (s, 3H), 3.44(m, 2H), 3.30 (d, 1H), 3.18 (dd, 1H), 3.07 (m, 3H), 2.81 (m, 2H),2.29-2.21 (m, 1H), 2.22 (s, 3H), 2.15 (s, 3H), 2.08 (s, 3H), 1.45 (s,9H), 1.44 (m, 1H).

¹³C-RMN (CDCl₃, 75 MHz) δ 169.95, 154.83, 148.77, 146.74, 145.06,144.19, 142.48, 141.30, 141.22, 138.49, 134.24, 131.47, 128.76, 127.46,127.33, 126.98, 126.76, 124.36, 124.09, 121.29, 121.08, 119.82, 119.62,118.03, 116.76, 116.41, 112.89, 112.44, 101.03, 79.65, 73.46, 60.38,58.76, 57.85, 56.21, 55.27, 51.76, 46.66, 41.49, 38.59, 34.48, 33.85,31.55, 28.37, 26.41, 24.29, 22.61, 15.51, 14.08, 9.53.

ESI-MS m/z: Calcd. for C₅₁H₅₇N₅O₈S: 899.4. Found (M+H)⁺: 900.4.

Example 75

To a solution of intermediate 69 (1.37 g, 1.52 mmol) in anhydrousacetonitrile (8 mL, 0.19 M) was added at 0° C. under Argon atmosphereDIPEA (5.31 mL, 30.4 mmol), MEMCl (2.59 mL, 22.8 mmol) and DMAP (18.63mg, 0.15 mmol). The reaction mixture was left at 23° C. under Argonatmosphere for 5 hours. A saturated aqueous solution of ammoniumchloride was added, the aqueous phase was extracted withdichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentethyl acetate/hexane 2:3) to afford intermediate 70 (1.38 g, 92%) as ayellow solid. Other fraction with 130 mg was isolated after column as amixture of starting material and compound 70 in a ratio 2:1.

R_(f): 0.48 (ethyl acetate/hexane 2:3)

¹H-RMN (CDCl₂, 300 MHz): δ 7.62 (d, 2H), 7.50 (d, 1H), 7.40 (t, 1H),7.33 (m, 2H), 7.21 (m, 2H), 6.55 (s, 1H), 6.08 (d, 1H), 5.97 (m, 1H),5.95-5.86 (m, 1H), 5.84 (d, 1H), 5.66 (broad d, 1H), 5.31 (dd, 1H), 5.21(dd, 1H), 5.14 (d, 1H), 5.04 (d, 1H), 4.16 (d, 1H), 4.06-3.92 (m, 3H),3.87 (m, 2H), 3.73 (m, 4H), 3.54 (m, 2H), 3.41. (s, 3H), 3.34 (s, 3H),3.30 (m, 2H), 3.20 (dd, 1H), 3.04 (m, 3H), 2.82 (m, 2H), 2.27 (m, 1H),2.24 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 1.49 (s, 9H), 1.43 (m, 1H).

¹³C-RMN (CDCl₃, 75 MHz): δ 170.19, 155.14, 148.96, 148.83, 148.31,145.37, 145.23, 144.43, 141.61, 141.55, 138.70, 134.42, 131.13, 131.02,127.76, 127.59, 127.23, 127.127.01, 125.58, 124.58, 124.27, 123.93,121.42, 120.09, 119.88, 118.29, 116.82, 113.03, 112.68, 101.31, 98.34,95.88, 92.55, 79.97, 73.50, 71.98, 71.88, 69.54, 67.68, 67.01, 59.73,59.27, 58.94, 58.27, 56.86, 56.35, 55.52, 51.90, 46.95, 41.63, 38.37,34.87, 33.67, 28.65, 26.70, 24.47, 15.83, 9.73.

ESI-MS m/z: Calcd. for C₅₅H₆₅N₅O₁₀S: 987.4. Found (M+1)⁺: 988.6.

Example 76

To a solution of intermediate 70 (1.38 g, 1.39 mmol) in anhydrousdichloromethane (36 mL, 0.04 M) was added at 23° C. under Argonatmosphere, (PPh₃)₂PdCl₂ (0.11 g, 8% in weight), acetic acid (0.39 mL,6.98 mmol) and tributyltin hydride (1.31 mL, 4.88 mmol). The reactionmixture was left at 23° C. under Argon atmosphere for 30 minutes,diluted with hexane and poured onto column (eluent mixtures ethylacetate/hexane in gradient from 0:100 to 3:2) to afford intermediate 71(1.16 g, 87%) as a yellow solid.

R_(f): 0.28 (ethyl acetate/hexane 1:1)

¹H-RMN (CDCl₃, 300 MHz): δ 7.69 (t, 2H), 7.54 (t, 2H), 7.39-7.23 (m 4H),6.61 (s, 1H), 5.98 (m, 1H), 5.96 (s, 1H), 5.87 (s, 1H), 5.80 (s, 1H),5.39 (t, 2H), 5.21 (d, 1H), 4.11 (m, 3H), 4.01 (m, 1H), 3.92 (m, 3H),3.66 (s, 3H), 3.55 (m, 2H), 3.39 (s, 3H), 3.37 (m, 3H), 3.26-3.12 (m,4H), 2.90 (d, 1H), 2.88 (m, 1H), 2.78 (d, 1H), 2.27 (s, 3H), 2.15 (s,3H), 2.06 (s, 3H), 1.73 (dd, 1H), 1.39 (s, 9H).

ESI-MS m/z: Calcd. for C₅₂H₆₁N₅O₁₀S: 947.4. Found (M+1)⁺: 948.8.

Example 77

To a solution of compound 71 (39 mg, 0.041 mmol) in anhydrous CH₂Cl₂(1.2 mL, 0.03M) was added at −10° C. under Argon atmosphere a solutionof benceneseleninic anhydride (21.14 mg, 0.041 mmol) in anhydrous CH₂Cl₂(0.6 mL). The reaction mixture was stirred at −10° C. under Argonatmosphere for 30 minutes. The reaction was diluted with CH₂Cl₂ andquenched with an aqueous saturated solution of sodium bicarbonate, theaqueous phase was extracted with CH₂Cl₂ and the organic layers weredried over sodium sulphate. The solvent was eliminated under reducedpressure and the crude of the reaction was purified by flash columnchromatography to afford compound 72 (33 mg, 83%) as a pale yellow solidand a mixture of isomers in ratio 1.3:1 by ¹H-RMN.

R_(f): 0.21 and 0.11 (ethyl acetate/hexane 2:1)

¹H-RMN (CDCl₃, 300 MHz) δ 7.74 (d, 4H), 7.70-7.62 (m, 4H), 7.39 (t, 4H),7.31 (t, 4H), 6.72 (m, 2H), 6.61 (s, 1H), 6.46 (s, 1H), 5.78 (s, 1H),5.77 (s, 1H), 5.61 (s, 1H), 5.58 (s, 1H), 5.38 (broad d, 1H), 5.23 (d,1H), 5.12 (d, 2H), 5.04 (d, 1H), 4.83 (s, 1H), 4.41 (s, 1H), 4.11 (m,2H), 4.03 (m, 4H), 3.90-3.86(m, 2H), 3.86 (s, 3H), 3.78-3.71 (m, 5H),3.55 (m, 6H), 3.52 (s, 3H), 3.38 (s, 3H), 3.35 (s, 3H), 3.27 (m, 4H),3.12 (m, 3H), 2.81 (m, 6H), 2.44 (m, 4H), 2.26 (s, 3H), 2.22 (s, 3H),2.14 (s, 3H), 2.14-2.10 (m, 2H), 2.04 (s, 3H), 1.78 (s, 3H), 1.77 (s,3H), 1.39 (s, 9H), 1.34 (s, 9H).

ESI-MS m/z: Calcd. for C₅₂H₆₁N₅O₁₁S: 963.4. Found (M+1)⁺: 964.9.

Example 78

The reaction flask was flamed twice, purged vacuum/Argon several timesand kept under Argon atmosphere for the reaction. To a solution of DMSO(220.8 μL) in anhydrous CH₂Cl₂ (20.7 mL) was dropwise added triflicanhydride (104.7 μL) at −78° C. The reaction mixture was stirred at −78°C. for 20 minutes, then a solution of 72 (300 mg, 0.31 mmol) inanhydrous CH₂Cl₂ (10.4 mL) at −78° C. was added via canula. During theaddition the temperature was kept at −78° C. in both flasks. Thereaction mixture was stirred at −40° C. for 35 minutes. After this time,^(i)Pr₂NEt (812.9 μL) was dropwise added and the reaction mixture waskept at 0° C. for 45 minutes. Then ^(t)BuOH (293.4 μL) and guanidine(534.9 μL) were dropwise added and the reaction mixture was stirred at23° C. for 40 minutes. After this time, acetic anhydride (441.1 μL) wasdropwise added and the reaction mixture was kept at 23° C. for 1 hourmore. Then the reaction mixture was diluted with CH₂Cl₂ and washed withan aqueous saturated solution of NH₄Cl, NaHCO₃ and NaCl. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated.

The residue was purified by a flash column chromatography (eluentmixtures of ethyl acetate/hexane in gradient from 1:4 to 1:1) to afford73 (160 mg, 64%) as a pale yellow solid.

R_(f): 0.13 (ethyl acetate/hexane 1:1)

¹H-RMN (300 MHz, CDCl₃): δ 6.76 (s, 1H), 6.06 (d, 5.98 (s, 1H), 5.32 (d,1H), 5.17 (d, 1H), 4.81 is, 1H), 4.48 (broad s, 1H), 4.36 (broad d, 1H),4.18 (s, 1H), 3.95-3.82 (m, 3H), 3.75 (s, 3H), 3.72-3.68 (m, 1H),3.59-3.52 (m, 4H), 3.37 (s, 3H), 3.36 (s, 3H), 2.96 (m, 2H), 2.56 (broadd, 1H), 2.29 (s, 3H), 2.28 (s, 3H), 2.19 (s, 3H), 1.98 (s, 3H), 1.69 (m,1H), 1.42-1.37 (m, 1H), 1.38 (s, 9H).

ESI-MS m/z: Calcd. for C₄₀H₅₁N₅O₁₁S: 809.3. Found (M+1)⁺: 810.2

Example 79

To a solution of intermediate 73 (169 mg, 0.21 mmol) in CHCl₃ (11 mL,0.02 M) was added at 23° C. p-TsOH (243 mg, 1.25 mmol). The reactionmixture was left at 23° C. and under Argon atmosphere for 14 hours. Thereaction was diluted with dichloromethane and a saturated solution ofsodium bicarbonate was added. The aqueous phase was extracted withdichloromethane, the combined organic layers were dried over sodiumsulphate, filtered and the solvent was eliminated under reducedpressure. The crude was purified by flash column chromatography (eluentmixtures of methylene chloride/methyl alcohol in gradient from 100:0 to9: 1) to afford intermediate 74 (123 mg, 95%) as a orange solid.

R_(f): 0.17 (methylene chloride/methyl alcohol 95:5)

¹H-RMN (300 MHz, CDCl₃): δ 6.81 (broad s, 1H), 6.49 (s, 1H), 6.06 (d,1H), 6.00 (d, 1H), 4.43 (broad s, 1H), 4.27 (d, 2H), 4.17 (s, 1H), 4.04(d, 1H), 3.77 (s, 3H), 3.62 (d, 1H), 3.39 (d, 1H), 3.18 (m, 1H), 3.00(dd, 2H), 2.65 (d, 1H), 2.57 (m, 1H), 2.33 (s, 3H), 2.27 (s, 3H), 2.17(s, 3H), 1.97 (s, 3H), 1.80 (d, 1H).

ESI-MS m/z: Calcd. for C₃₁H₃₅N₅O₇S: 621.2. Found (M+1)⁺: 622.2

Example 80

To a solution of the pyiridinium salt (285 mg, 1.14 mmol) in DMF (6 mL)was added at 23° C. a solution of intermediate 74 (71 mg, 0.114 mmol) indichloromethane (6 mL, 0.01 M final concentration). The reaction mixturewas left at 23° C. and under Argon atmosphere for 4 hours and 15minutes, then DBU (0.17 mL, 1.14 mmol) was added and the solution wasstirred at 23° C. and under Argon atmosphere for 15 minutes. After thistime a saturated solution of oxalic acid (11 mL) was added, and thereaction mixture was left at 23° C. under Argon atmosphere for 30minutes. The reaction mixture was cooled at 0° C., was diluted with Et₂Oand a saturated solution of sodium bicarbonate was added until to reachpH=5. The aqueous phase was extracted with Et₂O (×4) further basifiedwith more sodium bicarbonate and extracted with more Et₂O (×4). Thecombined organic layers were dried over sodium sulphate, filtered andthe solvent was eliminated under reduced pressure. The crude waspurified by flash column chromatography (eluent mixtures methylenechloride/methyl alcohol in gradient from 100:0 to 20:1) to affordintermediate 75 (38 mg, 55%) as a yellow solid.

R_(f): 0.7 and 0.5 (methylene chloride/methyl alcohol 8:1)

¹H-RMN (300 MHz, CDCl₃): δ (major isomer) 6.49 (s, 1H), 6.06 (d, 1H),6.01 (d, 1H), 5.78 (s, 1H), 4.55 (s, 1H), 4.37-4.23 (m, 3H), 4.05 (d,1H), 3.80 (s, 3H), 3.65 (d, 1H), 3.40 (broad d, 1H), 3.06-3.00 (m, 2H),2.66 (dd, 2H), 2.34 (s, 3H), 2.29 (s, 3H), 2.18 (s, 3H), 1.99 (s, 3H).

ESI-MS m/z: Calcd. for C₃₁H₃₂N₄O₈S: 620.2. Found (M+1)⁺: 621.1

Example 81

To a solution of intermediate 75 (23 mg, 0.037 mmol) in acetic acid (1.3mL) was added after 1 hour dopamine derivative (25 mg, 0.10 mmol). Thereaction mixture was left at 23° C. and under Argon atmosphere for 50hours. The solvent of the reaction was eliminated under reduced pressureand the residue was diluted with CH₂Cl₂, washed with an aqueoussaturated solution of sodium bicarbonate. The organic phase was driedover sodium sulphate, the solvent was eliminated under reduced pressureand the crude was purified by flash column chromatography (eluentmixtures of CH₂Cl₂/ethyl acetate in gradient from 100:0 to 1:2) toafford intermediate 76 (18 mg, 63%) as a pale yellow solid and a mixtureof isomers.

R_(f): 0.26 (CH₂Cl₂/ethyl acetate 1:3)

¹H-RMN (300 MHz, CDCl₃): δ 6.54 (s, 1H), 6.48 (d, 1H), 6.43 (s, 1H),6.08 (d, 1H), 6.06 (d, 1H), 6.04 (d, 1H), 6.01 (d, 1H), 5.80 (s, 1H),5.75 (s, 1H), 5.41 (m, 1H), 4.60-4.16 (m, 7H), 4.04 (m, 2H), 3.80 (s,3H), 3.79 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.65 (d, 2H), 3.42 (m,2H), 3.07-2.84 (m, 5H), 2.70 (d, 2H), 2.61-2.47 (m, 4H), 2.36 (s, 3H),2.34 (s, 3H), 2.31 (s, 3H), 2.19 (s, 3H), 2.16 (s, 3H), 2.04 (s, 3H),2.00 (s, 3H), 1.99 (s, 3H), 1.66 (m, 2H).

ESI-MS m/z: Calcd. for C₄₀H₄₃N₅O₉S: 769.3. Found (M+1)⁺: 770.0.

Example 82

To a solution of intermediate 76 (7 mg, 0.009 mmol) in acetonitrile (0.6mL) was added at 23° C. water (0.4 mL, 0.015 M, final concentration) andAgNO₃ (46 mg, 0.27 mmol). The reaction mixture was left under Argonatmosphere at 23° C. for 31 hours. The reaction was diluted withdichloromethane and a saturated solution of sodium bicarbonate and asaturated solution of sodium chloride was added. The aqueous phase wasextracted with dichloromethane and the combined organic layers weredried over sodium sulphate, filtered and the solvent was eliminatedunder reduced pressure. The crude was purified by flash columnchromatography (eluent dichloromethane/ethyl acetate in gradient from1/9 to 100% in ethyl acetate) to afford the final product 77 (4 mg, 58%)as a pale yellow solid.

R_(f): 0.17 (dichloromethane/ethyl acetate 1:9)

¹H-RMN (300 MHz, CDCl₃): δ 6.54 (s, 1H), 6.20 (m, 1H), 6.03 (d, 1H),6.01 (d, 1H), 5.72 (s, 1H), 5.41 (broad s, 1H), 4.65 (broad s, 1H),4.40-4.29 (m, 3H), 4.16 (d, 2H), 3.83 (m, 1H), 3.77 (s, 3H), 3.76 (s,3H), 3.49 (s, 1H), 3.21 (m, 2H), 2.94 (m, 4H), 2.66 (d, 1H), 2.51 (d,2H), 2.36 (m, 2H), 2.31 (s, 6H), 2.17 (s, 3H), 2.00 (s, 3H).

ESI-MS m/z: Calcd. for C₃₉H₄₄N₄O₁₀S: 760.3. Found (M−H₂O+1)⁺: 743.0

Bioassays for Antitumor Screening

The finality of these assays is to interrupt the growth of a “in vitro”tumor cell culture by means a continued exhibition of the cells to thesample to be testing.

Cell Lines Name N^(o) ATCC Species Tissue Characteristics P-388 CCL-46mouse ascites fluid lymphoid neoplasm K-562 CCL-243 human leukemiaerythroleukemia (pleural effusion) A-549 CCL-185 human lung lungcarcinoma “NSCL” SK-MEL-28 HTB-72 human melanoma malignant melanomaHT-29 HTB-38 human colon colon adenocarcinoma LoVo CCL-229 human coloncolon adenocarcinoma LoVo-Dox human colon colon adenocarcinoma (MDR)SW620 CCL-228 human colon colon adenocarcinoma (lymph node metastasis)DU-145 HTB-81 human prostate prostate carcinoma, not androgen receptorsLNCaP CRL-1740 human prostate prostate adenocarcinoma, with androgenreceptors SK-BR-3 HTB-30 human breast breast adenocarcinoma, Her2/neu+,(pleural effusion) MCF-7 HTB-22 human breast breast adenocarcinoma,(pleural effusion) MDA-MB-231 HTB-26 human breast breast adenocarcinoma,Her2/neu+, (pleural effusion) IGROV-1 human ovary ovary adenocarcinomaIGROV-ET human ovary ovary adenocarcinoma, characterized as ET-743resistant cells SK-OV-3 HTB-77 human ovary ovary adenocarcinoma(malignant ascites) OVCAR-3 HTB-161 human ovary ovary adenocarcinomaHeLa CCL-2 human cervix cervix epitheloid carcinoma HeLa-APL CCL-3 humancervix cervix epitheloid carcinoma, characterized as aplidine resistantcells A-498 HTB-44 human kidney kidney carcinoma PANC-1 CRL-1469 humanpancreas pancreatic epitheloid carcinoma HMEC1 human endothelium1°.-Inhibition of Cell Growth by Counting Cells.

This form of the assay employs 24 well multidishes of 16 mm diameter(Bergeron, 1984; Schroeder, 1981). The tumor cell lines employed are:P-388 (ATCC CCL 46), suspension culture of a lymphoid neoplasm from aDBA/2 mouse; A-549 (ATCC CCL 185), monolayer culture of a human lungcarcinoma; HT-29 (ATCC HTB-38), monolayer culture of a human coloncarcinoma; MEL-28 (ATCC HTB-72), monolayer culture of a human melanomaand DU-145 (ATCC HTB-81), monolayer culture of a human prostatecarcinoma.

Cells were maintained, in logarithmic phase of growth in Eagle's MinimumEssential Medium, with Earle's Balanced Salts, with non-essential aminoacids, with 2.0 mM L-Glutamine, without sodium bicarbonate (EMEM/neaa),supplemented with 10% Fetal Calf Serum (FCS), 10⁻² M. sodium bicarbonateand 0.1 U/1 penicillin G+0.1 g/l streptomycin sulfate. For theexperiments, cells are harvested from subconfluent cultures usingtrypsin and resuspended in fresh medium before plating.

P-388 cells were seeded into 16 mm diameter wells at 1×10⁴ cells perwell in 1 ml aliquots of EMEM 5% FCS containing different concentrationsof the sample to be tested. A separate set of cultures without drug wasseeded as control of growth, to ensure that cells remained inexponential phase of growth. All determinations are carrying out induplicate. After three days of incubation at 37° C., 5% CO₂ in a 98%humid atmosphere, an approximately IC50 was determined by comparing thegrowth in wells with drug to the growth in wells control.

A-549, HT-29, MEL-28 and DU-145 cells were seeded into 16 mm diameterwells at 1×10⁴ cells per well in 1 ml aliquots of EMEM 5% FCS containingdifferent concentrations of the sample to be tested. A separate set ofcultures without drug was seeded as control of growth, to ensure thatcells remained in exponential phase of growth. All determinations arecaring out in duplicate. After three days of incubation at 37° C., 5%CO₂ in a 98% humid atmosphere cells were stained with 0.1% crystalviolet. An approximately IC50 was determined by comparing the growth inwells with drug to the growth in wells control.

For quantifying the activity, after the incubation time, cells aretrypsinized and counted in a Coulter Counter ZM. All counts (net cellsper well), represent the average of duplicate wells. % G, percent ofgrowth relative to cultures without drug. The results of these assaysare used to generate dose-response curves from which more precise IC50values are determined (sample concentration which produces 50% cellgrowth inhibition).

Obtained results may predict the usefulness of a certain drug as apotential cancer treatment. For this technique, compounds which showIC50 values smaller than 1 μg/ml are selected to continue with furtherstudies. IC50's data allow to predict that not only if a drug could becystostatic, but also if it could have a potential in terms of tumorreduction.

2°.-Inhibition of Cells Growth by Colorimetric Assay.

A colorimetric type of assay, using sulforhodamine B (SRB) reaction hasbeen adapted for a quantitative measurement of cell growth and viability[following the technique described by Philip Skehan, et al. (1990), Newcalorimetric cytotoxicity assay for anticancer drug screening, J. Natl.Cancer Inst., 82:1107-1112]

This form of the assay employs 96 well cell culture microplates of 9 mmdiameter (Faircloth, 1988; Mosmann, 1983). Most of the cell lines areobtained from American Type Culture Collection (ATCC) derived fromdifferent human cancer types.

Cells are maintained in RPMI 1640 10% FBS, supplemented with 0.1 g/lpenicillin and 0.1 g/l streptomycin sulfate and then incubated at 37°C., 5% CO₂ and 98% humidity. For the experiments, cells were harvestedfrom subconfluent cultures using trypsin and resuspended in fresh mediumbefore plating.

Cells are seeded in 96 well microtiter plates, at 5×10³ cells per wellin aliquots of 195 μl medium, and they are allowed to attach to theplate surface by growing in drug free medium for 18 hours. Afterward,samples are added in aliquots of 5 μl in a ranging from 10 to 10⁻⁸μg/ml, dissolved in DMSO/EtOH/PBS (0.5:0.5:99). After 48 hours exposure,the antitumor effect are measured by the SRB methodology: cells arefixed by adding 50 μl of cold 50% (wt/vol) trichloroacetic acid (TCA)and incubating for 60 minutes at 4° C. Plates are washed with deionizedwater and dried. One hundred μl of SRB solution (0.4% wt/vol in 1%acetic acid) is added to each microtiter well and incubated for 10minutes at room temperature. Unbound SRB is removed by washing with 1%acetic acid. Plates are air dried and bound stain is solubilzed withTris buffer. Optical densities are read on a automatedspectrophotometric plate reader at a single wavelength of 490 nm.

The values for mean +/− SD of data from triplicate wells are calculated.Some parameters for cellular responses can be calculated: GI=growthinhibition, TGI=total growth inhibition (cytostatic effect) and LC=cellkilling (cytotoxic effect).

Obtained results may predict the usefulness of a certain drug as apotential cancer treatment. For this technique, compounds which showGI50 values smaller than 10 μg/ml are selected to continue with furtherstudies. GI50's data allow to predict that not only could a drug becystostatic, but also it could have a potential in terms of tumorreduction.

Activity Data (Molar) IC₅₀ 16 19 p388 1.63E−08 7.53E−10 a549 1.63E−087.53E−10 ht29 1.63E−08 7.53E−10 mel28 1.63E−08 7.53E−10 du145 7.53E−10

1 2 3 4 5 6 A549 GI₅₀ 1.53E−06 6.33E−07 4.18E−06 2.36E−06 7.21E−076.65E−06 TGI 6.07E−06 1.39E−06 1.30E−05 8.27E−06 2.40E−06 1.26E−05 LC₅₀1.92E−05 9.11E−06 1.32E−05 1.18E−05 8.41E−06 1.26E−05 HT29 GI₅₀ 1.11E−061.06E−06 4.01E−06 5.91E−07 4.81E−07 1.25E−05 TGI 1.92E−05 1.42E−051.23E−05 1.18E−06 1.20E−06 1.26E−05 LC₅₀ 1.92E−05 1.92E−05 1.32E−051.18E−05 6.01E−06 1.26E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ 5.61E−075.98E−07 TGI 1.15E−06 1.27E−06 LC₅₀ 6.07E−06 5.31E−06 OVCAR GI₅₀ TGILC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀ 5.67E−07 8.97E−07 TGI 1.07E−063.13E−06 LC₅₀ 1.92E−06 1.92E−05 MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGI LC₅₀H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 4.34E−07 5.92E−07 TGI 7.47E−07 1.11E−06LC₅₀ 1.29E−06 2.76E−06 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 9.01E−07 9.26E−07TGI 2.59E−06 2.26E−06 LC₅₀ 1.92E−05 1.82E−05 IGROV-ET GI₅₀ 9.70E−071.06E−06 TGI 1.92E−05 1.92E−05 LC₅₀ 1.92E−05 1.92E−05 SK-BR3 GI₅₀1.01E−06 7.78E−07 TGI 3.96E−06 1.75E−06 LC₅₀ 1.92E−05 1.92E−05 K562 GI₅₀3.61E−07 5.16E−07 TGI 7.99E−07 1.22E−06 LC₅₀ 1.77E−06 4.79E−06 PANC-1GI₅₀ 8.87E−07 8.90E−07 TGI 4.36E−06 3.26E−06 LC₅₀ 1.92E−05 1.92E−05 LOVOGI₅₀ 9.32E−07 5.33E−07 TGI 4.00E−06 1.07E−06 LC₅₀ 1.92E−05 1.92E−06LOVO-DOX GI₅₀ 6.17E−06 9.24E−07 TGI 1.92E−05 1.92E−05 LC₅₀ 1.92E−051.92E−05 HELA GI₅₀ 1.08E−06 7.63E−07 TGI 2.84E−06 1.45E−06 LC₅₀ 1.81E−059.80E−06 HELA-APL GI₅₀ 7.17E−07 4.79E−07 TGI 1.63E−06 8.44E−07 LC₅₀7.51E−06 1.49E−06 7 8 9 10 11 12 A549 GI₅₀ 1.87E−06 1.18E−05 1.64E−051.01E−03 1.19E−05 3.38E−07 TGI 3.51E−06 1.18E−05 1.64E−05 1.01E−031.19E−05 6.24E−07 LC₅₀ 6.61E−06 1.18E−05 1.64E−05 1.01E−03 1.19E−051.16E−06 HT29 GI₅₀ 9.18E−07 7.82E−06 1.64E−05 1.01E−03 1.19E−05 5.03E−07TGI 2.40E−06 1.18E−05 1.64E−05 1.01E−03 1.19E−05 1.27E−06 LC₅₀ 5.46E−061.18E−05 1.64E−05 1.01E−03 1.19E−05 1.54E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28GI₅₀ 1.19E−05 3.50E−07 TGI 1.19E−05 6.03E−07 LC₅₀ 1.19E−05 1.03E−06OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀ 1.19E−05 5.35E−07 TGI1.19E−05 1.02E−06 LC₅₀ 1.19E−05 4.22E−06 MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀TGI LC₅₀ H-MEC-1 GI₅₀ 2.15E−06 TGI 4.06E−06 LC₅₀ 7.63E−06 LNCAP GI₅₀5.84E−06 8.25E−08 TGI 1.19E−05 2.51E−07 LC₅₀ 1.19E−05 7.11E−07 SK-OV3GI₅₀ TGI LC₅₀ IGROV GI₅₀ 1.19E−05 2.25E−07 TGI 1.19E−05 4.86E−07 LC₅₀1.19E−05 1.05E−06 IGROV-ET GI₅₀ 1.19E−05 6.35E−07 TGI 1.19E−05 1.07E−06LC₅₀ 1.19E−05 3.82E−06 SK-BR3 GI₅₀ 1.19E−05 2.56E−07 TGI 1.19E−055.01E−07 LC₅₀ 1.19E−05 9.79E−07 K562 GI₅₀ 4.76E−06 1.10E−07 TGI 1.19E−052.51E−06 LC₅₀ 1.19E−05 1.54E−05 PANC-1 GI₅₀ 1.19E−05 4.72E−07 TGI1.19E−05 9.53E−07 LC₅₀ 1.19E−05 4.07E−06 LOVO GI₅₀ 1.19E−05 3.99E−07 TGI1.19E−05 8.08E−07 LC₅₀ 1.19E−05 3.21E−06 LOVO-DOX GI₅₀ 1.19E−05 5.04E−07TGI 1.19E−05 9.71E−07 LC₅₀ 1.19E−05 1.22E−05 HELA GI₅₀ 1.19E−05 3.44E−07TGI 1.19E−05 6.21E−07 LC₅₀ 1.19E−05 1.12E−06 HELA-APL GI₅₀ 1.19E−052.44E−07 TGI 1.19E−05 4.72E−07 LC₅₀ 1.19E−05 9.14E−07 13 14 15 17 18 20A549 GI₅₀ 3.94E−06 2.23E−06 2.00E−09 2.29E−08 1.61E−07 2.52E−08 TGI3.94E−06 2.23E−06 2.00E−09 2.29E−08 1.61−07 2.52E−08

HT29 GI₅₀ 6.02E−06 1.77E−06 4.84E−10 3.91E−08 8.04E−08 6.50E−09 TGI1.54E−05 6.39E−06 5.35E−09 8.60E−08 6.43E−07 5.36E−08 LC₅₀ 1.54E−051.25E−05 3.13E−06 1.27E−05 8.04E−05 6.67E−06 SW-620 GI₅₀ TGI LC₅₀ MEL-28GI₅₀ 2.45E−08 TGI 4.71E−08 LC₅₀ 9.11E−08 OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀TGI LC₅₀ DU145 GI₅₀ 1.59E−08 TGI 3.99E−08 LC₅₀ 1.47E−06 MCF GI₅₀ TGILC₅₀ MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ 4.74E−06 3.00E−06 5.56E−10 TGI1.54E−05 1.25E−05 1.32E−08 LC₅₀ 1.54E−05 1.25E−05 4.44E−06 LNCAP GI₅₀7.28E−09 TGI 2.28E−08 LC₅₀ 5.89E−08 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀2.59E−08 TGI 5.63E−08 LC₅₀ 1.22E−07 IGROV-ET GI₅₀ 2.77E−07 TGI 5.76E−07LC₅₀ 1.20E−06 SK-BR3 GI₅₀ 5.22E−09 TGI 1.45E−08 LC₅₀ 9.83E−08 K562 GI₅₀1.52E−09 TGI 9.11E−09 LC₅₀ 4.04E−07 PANC-1 GI₅₀ 3.71E−08 TGI 9.46E−08LC₅₀ 3.11E−06 LOVO GI₅₀ 2.87E−08 TGI 5.76E−08 LC₅₀ 1.15E−07 LOVO-DOXGI₅₀ 5.02E−07 TGI 1.83E−06 LC₅₀ 1.47E−05 HELA GI₅₀ 3.45E−09 TGI 7.09E−09LC₅₀ 1.46E−08 HELA-APL GI₅₀ 3.89E−09 TGI 8.09E−09 LC₅₀ 2.28E−08 21 22 2324 25 26 A549 GI₅₀ 3.45E−06 2.67E−08 6.06E−08 2.19E−08 9.27E−07 1.16E−05TGI 6.15E−06 5.90E−08 2.11E−07 4.62E−08 3.45E−06 1.16E−05 LC₅₀ 1.10E−051.31E−07 7.85E−07 9.74E−08 1.15E−05 1.16E−05 HT29 GI₅₀ 4.06E−06 2.97E−085.12E−08 5.17E−09 1.64E−06 1.16E−05 TGI 8.86E−06 8.25E−08 2.15E−071.39E−08 8.57E−06 1.16E−05 LC₅₀ 1.57E−05 1.31E−05 1.29E−05 1.45E−051.39E−05 1.16E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ 3.02E−06 3.02E−082.47E−07 2.01E−08 6.32E−07 1.16E−05 TGI 5.35E−06 5.55E−08 4.60E−074.27E−08 1.77E−06 1.16E−05 LC₅₀ 9.49E−06 1.02E−07 8.57E−07 9.09E−085.65E−06 1.16E−05 OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀3.40E−06 2.96E−08 5.85E−08 2.78E−08 8.61E−07 1.16E−05 TGI 6.07E−068.68E−08 1.43E−07 5.23E−08 2.20E−06 1.16E−05 LC₅₀ 1.09E−05 1.31E−064.45E−06 1.25E−07 6.57E−06 1.16E−05 MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGILC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 2.58E−06 6.15E−09 1.53E−078.40E−09 3.23E−07 3.82E−06 TGI 4.72E−06 2.11E−08 3.18E−07 2.53E−087.57E−07 1.16E−05 LC₅₀ 8.64E−06 5.50E−08 6.57E−07 6.44E−08 2.55E−061.16E−05 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 3.81E−06 1.91E−08 3.50E−078.21E−09 6.60E−07 6.84E−06 TGI 7.17E−06 4.46E−08 6.27E−07 3.18E−081.43E−06 1.16E−05 LC₅₀ 1.35E−05 1.04E−07 1.13E−06 1.10E−07 8.01E−061.16E−05 IGROV-ET GI₅₀ 3.56E−06 5.22E−08 4.56E−07 2.95E−08 1.61E−061.16E−05 TGI 6.51E−06 1.04E−07 7.60E−07 6.20E−08 4.41E−06 1.16E−05 LC₅₀1.19E−05 1.31E−07 1.26E−06 1.30E−07 1.21E−05 1.16E−05 SK-BR3 GI₅₀3.85E−06 5.77E−09 1.31E−08 2.48E−08 5.55E−07 4.23E−06 TGI 7.33E−062.23E−08 8.77E−08 6.38E−08 1.06E−06 9.20E−06 LC₅₀ 1.39E−05 1.20E−076.45E−07 2.69E−07 8.64E−06 1.16E−05 K562 GI₅₀ 3.52E−06 5.92E−09 2.03E−084.07E−09 3.16E−07 2.30E−06 TGI 7.76E−04 4.24E−08 7.33E−08 1.71E−087.75E−07 4.94E−06 LC₅₀ 1.57E−05 8.40E−07 1.40E−06 9.38E−08 3.78E−061.06E−05 PANC-1 GI₅₀ 3.21E−06 2.96E−08 5.09E−07 3.01E−08 1.37E−061.16E−05 TGI 6.62E−06 7.38E−08 1.26E−06 9.19E−08 3.90E−06 1.16E−05 LC₅₀1.37E−05 8.54E−07 1.29E−05 1.45E−05 1.10E−05 1.16E−05 LOVO GI₅₀ 3.30E−062.03E−08 2.76E−07 2.61E−08 9.38E−07 1.16E−05 TGI 6.20E−06 4.11E−085.22E−07 8.21E−08 3.16E−06 1.16E−05 LC₅₀ 1.16E−05 9.65E−08 9.83E−071.45E−05 9.27E−06 1.16E−05 LOVO-DOX GI₅₀ 4.88E−06 8.74E−08 6.61E−071.15E−07 9.04E−07 1.16E−05 TGI 1.24E−05 5.24E−07 7.46E−06 1.74E−063.77E−06 1.16E−05 LC₅₀ 1.57E−05 1.31E−05 1.29E−05 1.45E−05 1.39E−051.16E−05 HELA GI₅₀ 3.40E−06 3.32E−08 3.82E−08 2.03E−08 1.50E−06 1.16E−05TGI 5.90E−06 5.66E−08 6.60E−08 4.76E−08 3.48E−06 1.16E−05 LC₅₀ 1.02E−051.58E−07 1.14E−07 1.12E−07 8.11E−06 1.16E−05 HELA-APL GI₅₀ 3.24E−062.11E−08 3.56E−08 2.03E−08 9.39E−07 1.16E−05 TGI 5.98E−06 4.44E−086.03E−08 4.65E−08 2.78E−06 1.16E−05 LC₅₀ 1.11E−05 9.35E−08 1.02E−071.07E−07 7.69E−06 1.16E−05 27 28 29 30 31 32 A549 GI₅₀ 6.26E−08 5.25E−084.80E−06 3.47E−09 1.83E−09 2.38E−06 TGI 2.06E−07 1.63E−07 1.14E−051.05E−08 5.97E−09 4.29E−06 LC₅₀ 1.89E−06 2.27E−06 1.14E−05 8.90E−089.21E−08 7.70E−06 HT29 GI₅₀ 7.56E−08 7.65E−08 1.14E−05 2.03E−09 1.97E−093.48E−06 TGI 1.42E−06 2.05E−06 1.14E−05 1.35E−08 1.62E−08 6.10E−06 LC₅₀1.42E−05 1.22E−05 1.14E−05 1.54E−05 3.68E−06 1.07E−05 SW-620 GI₅₀ TGILC₅₀ MEL-28 GI₅₀ 6.14E−08 5.68E−08 3.74E−06 1.09E−09 8.32E−10 2.41E−06TGI 1.34E−07 1.35E−07 1.14E−05 3.66E−09 2.54E−09 4.14E−06 LC₅₀ 5.94E−076.92E−07 1.14E−05 1.07E−08 8.06E−09 7.13E−06 OVCAR GI₅₀ TGI LC₅₀ A498GI₅₀ TGI LC₅₀ DU145 GI₅₀ 7.55E−08 3.58E−08 2.30E−06 2.63E−09 4.44E−102.48E−06 TGI 8.48E−07 1.27E−06 7.93E−06 7.44E−09 9.51E−10 4.83E−06 LC₅₀1.42E−05 1.22E−05 1.14E−05 1.41E−05 2.23E−06 9.39E−06 MCF GI₅₀ TGI LC₅₀MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 2.61E−08 2.29E−088.78E−07 9.68E−08 4.19E−10 1.77E−06 TGI 6.46E−08 5.04E−08 6.07E−061.20E−07 1.63E−08 3.36E−06 LC₅₀ 2.61E−07 1.11E−07 1.14E−05 1.47E−079.07E−08 6.41E−06 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 4.07E−08 4.90E−082.19E−06 6.95E−10 4.13E−10 1.97E−06 TGI 1.02E−07 1.55E−07 1.14E−053.32E−09 1.07E−09 3.91E−06 LC₅₀ 1.02E−06 1.57E−06 1.14E−05 2.31E−071.26E−08 7.74E−06 IGROV-ET GI₅₀ 1.65E−07 1.38E−07 3.12E−06 2.54E−082.91E−09 2.85E−06 TGI 3.87E−07 3.52E−07 1.14E−05 8.24E−08 7.49E−095.24E−06 LC₅₀ 9.15E−07 8.98E−07 1.14E−05 1.54E−05 2.09E−06 9.67E−06SK-BR3 GI₅₀ 5.58E−08 4.73E−08 8.89E−07 5.27E−10 3.96E−10 2.63E−07 TGI1.97E−07 1.64E−07 7.98E−06 2.29E−09 1.22E−09 1.66E−06 LC₅₀ 1.05E−061.22E−06 1.14E−05 2.67E−07 6.07E−08 7.52E−06 K562 GI₅₀ 2.17E−08 2.83E−086.29E−07 3.12E−10 2.26E−10 6.94E−07 TGI 5.66E−08 1.35E−07 3.90E−062.49E−09 7.92E−10 2.20E−06 LC₅₀ 2.06E−07 2.69E−06 1.14E−05 9.51E−061.26E−07 6.81E−06 PANC-1 GI₅₀ 9.28E−08 5.92E−08 6.55E−06 2.89E−098.07E−10 2.56E−06 TGI 1.45E−06 1.97E−07 1.14E−05 1.55E−08 6.68E−094.75E−06 LC₅₀ 1.42E−05 1.22E−05 1.14E−05 1.54E−05 1.77E−06 8.79E−06 LOVOGI₅₀ 8.04E−08 6.63E−08 2.85E−06 2.00E−09 6.88E−10 1.89E−06 TGI 2.36E−072.12E−07 1.14E−05 8.07E−09 3.49E−09 3.76E−06 LC₅₀ 7.07E−07 6.87E−071.14E−05 1.54E−05 1.82E−06 7.39E−06 LOVO-DOX GI₅₀ 4.31E−07 3.62E−074.09E−06 2.07E−07 9.05E−09 8.02E−07 TGI 1.42E−05 1.22E−06 1.14E−052.07E−06 1.48E−06 2.48E−06 LC₅₀ 1.42E−05 1.22E−05 1.14E−05 1.54E−057.21E−06 6.76E−06 HELA GI₅₀ 9.78E−08 3.86E−08 6.30E−06 1.15E−09 6.78E−102.53E−07 TGI 2.75E−07 7.55E−08 1.14E−05 4.53E−09 2.04E−09 4.18E−07 LC₅₀7.86E−07 2.89E−07 1.14E−05 2.17E−08 1.12E−08 6.89E−07 HELA-APL GI₅₀9.51E−08 5.59E−08 4.29E−06 1.86E−09 5.76E−10 3.39E−08 TGI 3.04E−071.30E−07 1.14E−05 5.50E−09 1.96E−09 6.05E−08 LC₅₀ 9.51E−07 6.82E−071.14E−05 1.95E−08 9.96E−09 1.08E−07 33 34 35 36 37 38 A549 GI₅₀ 2.62E−085.53E−08 2.93E−06 5.56E−09 2.64E−08 4.73E−07 TGI 6.24E−08 1.84E−075.96E−06 2.29E−08 7.13E−08 1.42E−06 LC₅₀ 1.49E−07 6.11E−07 1.21E−058.38E−08 8.75E−07 4.71E−06 HT29 GI₅₀ 1.73E−08 6.20E−08 6.02E−07 2.73E−092.90E−08 6.53E−07 TGI 2.71E−07 1.65E−07 8.18E−06 1.17E−08 2.62E−071.67E−06 LC₅₀ 9.81E−06 1.64E−06 1.44E−05 1.56E−05 1.05E−05 4.73E−06SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ 1.28E−08 3.27E−08 3.90E−07 1.84E−091.41E−08 2.72E−07 TGI 3.67E−08 6.03E−08 8.63E−07 4.01E−09 3.42E−084.83E−07 LC₅₀ 9.79E−08 1.11E−07 3.19E−06 8.71E−09 8.21E−08 8.60E−07OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀ 8.02E−09 5.11E−085.70E−07 3.58E−09 7.50E−09 2.86E−07 TGI 1.56E−08 9.87E−08 1.20E−069.55E−09 1.48E−08 5.64E−07 LC₅₀ 4.41E−06 5.37E−08 1.03E−05 1.04E−054.43E−06 1.11E−06 MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGILC₅₀ LNCAP GI₅₀ 2.86E−09 2.20E−08 1.89E−07 4.60E−10 2.86E−09 3.38E−07TGI 5.64E−09 5.21E−08 3.97E−07 1.37E−09 5.26E−09 5.18E−07 LC₅₀ 1.11E−081.47E−07 8.34E−07 5.06E−09 9.67E−09 7.96E−07 SK-OV3 GI₅₀ TGI LC₅₀ IGROVGI₅₀ 9.85E−08 2.92E−08 4.20E−07 1.12E−09 7.24E−09 3.81E−07 TGI 4.26E−075.83E−08 1.69E−06 3.79E−09 2.63E−08 7.86E−07 LC₅₀ 1.58E−06 1.17E−078.63E−06 1.04E−08 1.81E−06 4.18E−06 IGROV-ET GI₅₀ 8.95E−08 2.30E−062.77E−08 2.99E−08 3.65E−07 TGI 2.62E−07 6.35E−06 7.17E−08 1.04E−076.75E−07 LC₅₀ 8.60E−07 1.44E−05 1.56E−05 5.71E−06 1.48E−06 SK-BR3 GI₅₀4.83E−09 1.27E−08 3.96E−07 2.87E−09 4.15E−09 1.94E−07 TGI 1.39E−087.01E−08 1.10E−06 6.65E−09 1.01E−08 4.57E−07 LC₅₀ 3.85E−07 6.26E−079.35E−06 1.54E−08 4.01E−07 1.07E−06 K562 GI₅₀ 5.49E−09 1.62E−08 1.93E−072.46E−10 5.05E−09 1.93E−07 TGI 1.59E−07 4.50E−08 1.13E−06 7.00E−101.37E−08 5.10E−07 LC₅₀ 2.83E−06 1.46E−07 9.77E−06 5.11E−09 7.37E−071.54E−06 PANC-1 GI₅₀ 1.99E−08 5.11E−08 4.71E−07 2.82E−09 3.09E−086.16E−07 TGI 1.22E−07 1.23E−07 1.41E−06 9.72E−09 9.57E−08 1.38E−06 LC₅₀4.51E−06 8.41E−07 1.44E−05 2.60E−06 2.69E−06 4.50E−06 LOVO GI₅₀ 1.99E−082.87E−08 5.64E−07 2.12E−09 1.62E−08 3.58E−07 TGI 5.36E−08 5.32E−082.14E−06 5.91E−09 1.18E−07 7.93E−07 LC₅₀ 1.45E−07 9.86E−08 1.44E−051.56E−08 6.40E−06 2.51E−06 LOVO-DOX GI₅₀ 3.67E−07 1.40E−07 5.75E−062.46E−07 8.68E−08 1.54E−06 TGI 9.40E−07 4.10E−07 1.44E−05 1.09E−068.97E−07 3.08E−06 LC₅₀ 5.90E−06 1.20E−05 1.44E−05 1.56E−05 6.96E−066.15E−06 HELA GI₅₀ 5.02E−09 3.35E−08 3.96E−07 1.46E−09 4.04E−09 3.17E−07TGI 1.61E−08 6.24E−08 8.37E−07 3.85E−09 8.55E−09 5.45E−07 LC₅₀ 9.69E−081.16E−07 3.39E−06 9.82E−09 4.73E−08 9.35E−07 HELA-APL GI₅₀ 5.24E−093.39E−08 4.58E−07 1.85E−09 7.10E−09 2.95E−07 TGI 1.91E−08 6.05E−081.09E−06 4.30E−09 2.06E−08 5.49E−07 LC₅₀ 1.15E−07 1.08E−07 5.92E−061.00E−08 7.70E−08 1.02E−06 39 40 41 42 43 44 A549 GI₅₀ 8.49E−09 1.95E−074.31E−08 5.74E−08 2.61E−08 2.74E−08 TGI 4.51E−08 4.47E−07 7.35E−081.47E−07 6.21E−08 5.67E−08 LC₅₀ 1.49E−06 1.03E−06 1.25E−07 2.37E−061.48E−07 4.70E−07 HT29 GI₅₀ 5.34E−09 3.37E−07 4.27E−08 3.97E−08 2.42E−083.88E−08 TGI 1.97E−07 1.05E−06 1.20E−07 1.31E−07 1.90E−07 1.54E−06 LC₅₀3.77E−06 3.84E−06 1.47E−05 1.65E−05 1.67E−05 1.11E−05 SW-620 GI₅₀ TGILC₅₀ MEL-28 GI₅₀ 3.03E−09 1.62E−07 3.37E−08 3.01E−08 9.09E−09 2.34E−08TGI 5.43E−09 3.34E−07 6.45E−08 5.68E−08 3.27E−08 4.59E−08 LC₅₀ 9.76E−096.88E−07 1.23E−07 1.07E−07 9.10E−08 9.01E−08 OVCAR GI₅₀ TGI LC₅₀ A498GI₅₀ TGI LC₅₀ DU145 GI₅₀ 3.52E−09 5.63E−06 4.53E−08 3.74E−08 1.36E−082.34E−08 TGI 9.85E−09 1.60E−07 7.55E−08 8.25E−08 6.70E−08 7.19E−08 LC₅₀2.01E−06 1.63E−06 2.74E−07 2.47E−07 2.92E−06 1.11E−05 MCF GI₅₀ TGI LC₅₀MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 7.35E−10 4.81E−089.50E−40 5.96E−09 6.58E−09 4.53E−09 TGI 2.49E−09 1.62E−07 3.14E−082.24E−08 2.02E−08 2.04E−08 LC₅₀ 6.73E−09 5.09E−07 9.28E−08 6.37E−088.87E−08 4.80E−08 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 3.13E−09 9.13E−081.57E−08 2.86E−08 1.06E−08 3.26E−08 TGI 7.47E−09 3.17E−07 4.37E−086.63E−08 4.96E−08 7.80E−08 LC₅₀ 5.26E−07 1.28E−06 1.21E−07 1.53E−073.84E−07 1.11E−05 IGROV-ET GI₅₀ 4.19E−08 2.89E−07 4.18E−08 2.80E−076.85E−08 4.92E−08 TGI 8.86E−08 5.33E−07 8.71E−08 1.02E−06 3.69E−074.43E−07 LC₅₀ 2.44E−06 9.80E−07 1.47E−05 1.65E−05 5.80E−06 1.11E−05SK-BR3 GI₅₀ 2.68E−09 4.26E−08 3.23E−08 5.83E−09 3.27E−09 TGI 6.98E−099.76E−08 7.22E−08 2.42E−08 1.19E−08 LC₅₀ 5.57E−08 5.24E−07 3.52E−071.64E−07 7.61E−08 K562 GI₅₀ 1.06E−09 4.14E−08 2.04E−08 1.78E−08 3.11E−097.97E−09 TGI 5.58E−09 1.31E−07 5.05E−08 5.96E−08 1.38E−08 7.79E−08 LC₅₀6.98E−07 7.76E−07 1.25E−07 6.94E−07 8.95E−07 1.11E−05 PANC-1 GI₅₀4.91E−09 3.21E−07 3.77E−08 3.90E−08 2.29E−08 7.52E−08 TGI 1.47E−087.47E−07 1.00E−07 1.15E−08 8.27E−08 3.44E−07 LC₅₀ 2.36E−06 2.57E−061.47E−05 5.27E−06 2.15E−06 1.11E−05 LOVO GI₅₀ 3.72E−09 2.13E−07 3.78E−083.77E−08 3.12E−08 3.39E−08 TGI 9.00E−09 5.52E−07 9.40E−08 7.26E−088.67E−08 1.43E−07 LC₅₀ 9.71E−07 1.68E−06 1.47E−05 2.93E−06 1.67E−051.11E−05 LOVO-DOX GI₅₀ 3.26E−07 3.54E−07 2.70E−07 5.15E−07 4.23E−072.97E−07 TGI 9.12E−07 8.10E−07 1.18E−06 1.44E−06 1.15E−06 9.19E−07 LC₅₀4.12E−06 2.79E−06 1.47E−05 1.65E−05 1.67E−05 1.11E−05 HELA GI₅₀ 2.84E−091.11E−07 2.82E−08 2.81E−08 9.39E−09 2.02E−08 TGI 5.69E−09 2.76E−075.81E−08 6.25E−08 3.99E−08 4.20E−08 LC₅₀ 1.14E−08 6.61E−07 1.20E−071.39E−07 1.45E−07 8.75E−08 HELA-APL GI₅₀ 2.76E−09 1.53E−07 3.01E−083.46E−08 2.41E−08 2.50E−08 TGI 5.20E−09 3.44E−07 6.31E−08 6.70E−087.32E−08 5.96E−08 LC₅₀ 9.82E−09 7.75E−07 1.32E−07 1.30E−07 4.23E−073.52E−07 45 46 47 48 49 50 A549 GI₅₀ 1.62E−07 2.89E−08 1.02E−05 8.10E−061.84E−07 3.76E−06 TGI 4.13E−07 6.18E−08 1.02E−05 1.02E−05 4.31E−071.13E−05 LC₅₀ 1.50E−06 2.77E−06 1.02E−05 1.02E−05 1.01E−06 1.13E−05 HT29GI₅₀ 8.36E−08 4.29E−08 1.02E−05 6.71E−06 1.94E−07 8.90E−06 TGI 1.41E−061.17E−07 1.02E−05 1.02E−05 5.88E−07 1.13E−05 LC₅₀ 1.00E−05 1.17E−051.02E−05 1.02E−05 1.13E−05 1.13E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀1.17E−07 2.96E−08 8.70E−06 3.19E−06 1.81E−07 2.53E−06 TGI 2.85E−075.64E−08 1.02E−05 1.02E−05 3.56E−07 8.36E−06 LC₅₀ 6.89E−07 1.07E−071.02E−05 1.02E−05 7.01E−07 1.13E−05 OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGILC₅₀ DU145 GI₅₀ 3.59E−08 2.34E−08 4.12E−06 2.55E−06 1.07E−07 1.80E−06TGI 1.03E−07 4.96E−08 1.02E−05 6.26E−06 1.25E−06 9.18E−04 LC₅₀ 1.00E−051.05E−07 1.02E−05 1.02E−05 1.13E−05 1.13E−03 MCF GI₅₀ TGI LC₅₀ MB231GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 3.83E−09 1.47E−084.49E−07 1.67E−06 4.05E−08 9.48E−07 TGI 1.73E−08 2.96E−08 2.92E−063.24E−06 1.75E−07 3.68E−06 LC₅₀ 5.15E−08 5.92E−08 1.02E−05 6.31E−064.42E−07 1.13E−05 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 5.69E−08 2.99E−084.22E−06 3.52E−06 1.65E−07 2.42E−06 TGI 3.92E−07 7.24E−08 1.02E−051.02E−05 4.32E−07 1.13E−05 LC₅₀ 1.00E−05 1.17E−05 1.02E−05 1.02E−051.13E−06 1.13E−03 IGROV-ET GI₅₀ 8.65E−08 2.85E−07 4.92E−06 3.72E−062.80E−07 3.29E−06 TGI 4.97E−07 1.35E−06 1.02E−05 1.02E−05 6.16E−071.13E−05 LC₅₀ 1.00E−05 1.17E−05 1.02E−05 1.02E−05 1.13E−05 1.13E−05SK-BR3 GI₅₀ 2.83E−09 1.86E−08 3.01E−07 6.82E−07 4.96E−08 1.31E−05 TGI7.56E−09 5.32E−08 1.37E−06 4.00E−06 1.86E−06 1.13E−05 LC₅₀ 1.33E−072.47E−07 1.02E−05 1.02E−05 1.13E−05 1.13E−05 K562 GI₅₀ 1.76E−08 2.30E−081.73E−07 5.15E−07 3.77E−08 8.91E−07 TGI 1.50E−07 6.48E−08 2.32E−062.73E−06 3.11E−07 1.13E−05 LC₅₀ 1.00E−05 4.25E−06 1.02E−05 1.02E−055.10E−06 1.13E−05 PANC-1 GI₅₀ 2.29E−07 6.19E−08 1.02E−05 4.42E−061.79E−07 2.45E−06 TGI 1.24E−06 3.98E−07 1.02E−05 1.02E−05 5.64E−071.13E−05 LC₅₀ 1.00E−05 1.17E−05 1.02E−05 1.02E−05 3.92E−06 1.13E−05 LOVOGI₅₀ 1.30E−07 5.53E−08 9.16E−06 4.01E−06 2.50E−07 4.94E−06 TGI 2.83E−073.04E−07 1.02E−05 1.02E−05 4.71E−07 1.13E−05 LC₅₀ 6.14E−07 1.17E−051.02E−05 1.02E−05 8.90E−07 1.13E−05 LOVO-DOX GI₅₀ 4.79E−07 4.26E−071.02E−05 1.02E−05 6.72E−07 1.01E−05 TGI 3.14E−06 1.35E−06 1.02E−051.02E−05 1.13E−05 1.13E−05 LC₅₀ 1.00E−05 1.17E−05 1.02E−05 1.02E−051.13E−05 1.13E−05 HELA GI₅₀ 1.64E−08 2.64E−08 4.10E−06 2.17E−06 1.89E−073.75E−06 TGI 3.62E−08 5.01E−08 1.02E−05 5.11E−06 4.18E−07 1.13E−05 LC₅₀7.97E−08 9.74E−08 1.02E−05 1.02E−05 9.29E−07 1.13E−05 HELA-APL GI₅₀2.84E−08 2.77E−08 3.74E−06 1.87E−06 1.82E−07 3.11E−06 TGI 1.21E−075.20E−08 1.02E−05 3.83E−06 3.57E−07 1.13E−05 LC₅₀ 7.83E−07 9.78E−081.02E−05 7.87E−06 7.01E−07 1.13E−05 51 52 53 54 55 56 A549 GI₅₀ 2.85E−098.01E−09 3.34E−09 2.02E−08 6.84E−08 3.02E−08 TGI 6.59E−09 5.78E−088.17E−09 6.63E−08 2.86E−07 6.87E−08 LC₅₀ 5.42E−07 9.73E−07 2.21E−066.36E−07 1.22E−06 7.05E−07 HT29 GI₅₀ 5.24E−09 6.14E−09 4.33E−09 2.99E−087.57E−08 4.88E−08 TGI 2.37E−06 1.25E−06 1.83E−08 1.23E−07 1.81E−065.71E−07 LC₅₀ 1.18E−08 7.78E−06 2.74E−06 1.20E−05 1.27E−05 1.20E−05SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ 2.71E−09 4.07E−09 2.21E−09 1.55E−088.86E−08 2.63E−08 TGI 4.64E−09 1.55E−08 4.00E−09 3.61E−08 2.65E−074.90E−08 LC₅₀ 7.94E−09 5.27E−08 7.20E−09 8.39E−08 6.98E−07 9.14E−08OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀ 5.24E−09 4.92E−093.58E−09 3.76E−09 5.90E−08 4.10E−08

MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀2.31E−09 2.39E−09 1.87E−09 7.42E−09 1.71E−08 1.22E−08 TGI 3.96E−094.66E−09 3.95E−09 2.93E−08 3.60E−08 2.88E−08 LC₅₀ 6.82E−09 9.06E−098.33E−09 1.09E−07 7.57E−08 6.77E−08 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀3.35E−09 7.32E−09 2.74E−09 6.56E−09 6.92E−08 3.42E−08 TGI 6.45E−094.33E−08 6.72E−09 3.94E−08 2.71E−07 6.93E−08 LC₅₀ 1.18E−04 3.06E−062.74E−06 3.37E−06 1.58E−06 1.06E−06 IGROV-ET GI₅₀ 4.36E−08 3.95E−081.02E−08 3.70E−08 2.48E−07 4.02E−08 TGI 1.18E−07 1.19E−07 1.40E−061.20E−07 6.24E−07 7.97E−08 LC₅₀ 1.18E−05 8.53E−06 6.45E−06 1.20E−054.18E−06 1.20E−05 SK-BR3 GI₅₀ 5.05E−09 6.18E−09 2.95E−09 1.27E−085.71E−09 TGI 3.44E−07 2.58E−07 2.99E−08 1.54E−07 1.86E−08 LC₅₀ 1.18E−051.19E−05 1.82E−06 1.20E−05 4.22E−07 K562 GI₅₀ 3.15E−09 4.51E−09 1.54E−091.88E−09 5.84E−08 6.89E−09 TGI 7.75E−09 2.13E−08 1.30E−08 5.18E−092.72E−07 8.68E−08 LC₅₀ 6.53E−07 8.06E−08 5.83E−07 2.70E−06 1.04E−061.20E−05 PANC-1 GI₅₀ 3.53E−09 2.15E−08 3.36E−09 2.30E−08 1.48E−079.02E−08 TGI 7.73E−09 6.24E−08 1.01E−08 6.48E−08 1.90E−06 9.53E−07 LC₅₀5.20E−07 6.10E−07 1.44E−06 5.31E−06 1.27E−05 1.20E−05 LOVO GI₅₀ 3.93E−099.17E−09 3.82E−09 3.85E−09 1.51E−07 2.30E−08 TGI 8.86E−09 2.96E−088.54E−09 1.44E−08 3.56E−07 4.82E−08 LC₅₀ 1.18E−05 8.57E−08 2.94E−061.20E−05 8.37E−07 1.01E−07 LOVO-DOX GI₅₀ 3.52E−07 2.73E−07 5.79E−083.38E−07 3.33E−07 3.33E−07 TGI 1.18E−05 1.19E−05 1.45E−06 1.20E−051.57E−06 1.33E−06 LC₅₀ 1.18E−05 1.19E−05 5.24E−06 1.20E−05 1.27E−051.20E−05 HELA GI₅₀ 3.43E−09 5.98E−09 3.12E−09 2.19E−08 1.48E−07 2.71E−08TGI 6.63E−09 2.25E−08 7.55E−09 6.41E−08 3.63E−07 5.31E−08 LC₅₀ 1.55E−081.85E−06 8.39E−08 3.76E−07 8.87E−07 1.04E−07 HELA-APL GI₅₀ 3.49E−097.05E−09 2.99E−09 1.33E−08 1.11E−07 2.59E−08 TGI 6.18E−09 2.14E−081.22E−08 1.43E−07 3.23E−07 5.83E−08 LC₅₀ 1.09E−08 9.23E−08 3.89E−084.59E−07 9.15E−07 2.04E−07 57 58 59 60 61 62 A549 GI₅₀ 3.74E−08 2.16E−071.59E−08 3.60E−09 2.43E−08 4.56E−09 TGI 1.01E−07 5.17E−07 4.85E−081.31E−08 5.01E−08 1.65E−08 LC₅₀ 1.50E−06 1.24E−06 2.73E−07 7.60E−081.04E−07 3.34E−07 HT29 GI₅₀ 4.46E−08 2.97E−07 3.83E−09 2.28E−09 6.58E−094.03E−09 TGI 4.38E−06 1.54E−06 1.60E−08 1.31E−08 1.40E−06 2.83E−07 LC₅₀1.22E−05 1.24E−05 4.28E−06 1.28E−05 1.31E−05 1.25E−05 SW-620 GI₅₀ TGILC₅₀ MEL-28 GI₅₀ 2.93E−08 2.41E−07 3.56E−09 1.91E−09 6.19E−09 2.46E−09TGI 5.56E−08 4.69E−07 1.10E−08 4.27E−09 1.88E−08 4.92E−09 LC₅₀ 1.06E−079.69E−07 3.04E−06 9.55E−09 6.07E−08 9.78E−09 OVCAR GI₅₀ TGI LC₅₀ A498GI₅₀ TGI LC₅₀ DU145 GI₅₀ 5.21E−08 4.68E−08 4.09E−09 1.80E−09 5.14E−093.30E−09 TGI 1.22E−05 8.88E−08 1.02E−08 1.36E−06 9.34E−09 1.04E−08 LC₅₀1.22E−05 1.24E−05 1.64E−06 7.13E−06 7.97E−06 3.59E−06 MCF GI₅₀ TGI LC₅₀MB231 GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 1.51E−08 2.99E−081.03E−09 4.15E−10 1.17E−09 3.01E−09 TGI 3.23E−08 7.16E−08 2.67E−091.42E−09 2.94E−09 5.03E−09 LC₅₀ 6.93E−08 2.40E−07 6.43E−09 5.03E−096.92E−09 8.40E−09 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 2.88E−08 9.39E−083.01E−09 1.38E−09 4.82E−09 2.59E−09 TGI 6.60E−08 3.05E−07 7.33E−094.03E−09 9.64E−09 6.55E−09 LC₅₀ 6.48E−06 1.04E−06 1.10E−07 1.18E−085.25E−07 3.90E−06 IGROV-ET GI₅₀ 2.52E−07 1.88E−07 4.48E−09 2.09E−082.25E−07 2.66E−08 TGI 5.44E−07 4.22E−07 1.07E−07 5.31E−08 7.12E−076.95E−08 LC₅₀ 1.17E−06 9.49E−07 3.04E−06 2.89E−06 4.77E−06 2.92E−06SK-BR3 GI₅₀ 6.42E−09 2.09E−08 4.31E−10 4.28E−09 1.01E−09 TGI 3.18E−087.50E−08 1.22E−09 9.00E−09 4.74E−09 LC₅₀ 3.82E−07 2.55E−06 4.22E−082.45E−07 1.03E−07 K562 GI₅₀ 8.82E−09 2.64E−08 1.80E−09 8.09E−10 2.56E−091.94E−09 TGI 8.88E−08 9.42E−08 9.83E−09 1.32E−08 8.62E−09 1.10E−08 LC₅₀1.22E−05 1.24E−05 3.29E−06 8.57E−06 6.42E−07 6.02E−06 PANC-1 GI₅₀5.29E−08 3.79E−07 4.76E−09 3.31E−09 4.69E−09 3.86E−09 TGI 1.34E−061.27E−06 4.23E−08 1.19E−08 1.54E−08 1.35E−08 LC₅₀ 1.22E−05 1.24E−053.75E−06 1.28E−05 1.31E−05 4.75E−06 LOVO GI₅₀ 8.59E−08 1.39E−07 5.80E−093.27E−09 1.71E−08 5.03E−09 TGI 2.74E−07 2.85E−07 2.33E−08 8.23E−097.09E−08 1.79E−08 LC₅₀ 8.77E−07 5.81E−07 1.19E−06 2.90E−06 8.23E−061.54E−06 LOVO-DOX GI₅₀ 7.74E−07 4.83E−07 1.23E−07 1.63E−07 4.95E−071.36E−07 TGI 6.22E−06 2.26E−06 7.09E−07 7.57E−07 1.76E−06 5.58E−07 LC₅₀1.22E−05 1.24E−05 3.93E−06 1.28E−05 1.31E−05 1.31E−05 HELA GI₅₀ 2.68E−083.94E−08 2.44E−09 1.03E−09 3.56E−09 2.37E−09 TGI 5.64E−08 7.08E−085.08E−09 3.41E−09 6.49E−09 5.11E−09 LC₅₀ 1.12E−07 1.65E−07 1.06E−081.28E−08 1.18E−08 1.10E−08 HELA-APL GI₅₀ 2.23E−08 6.10E−08 3.32E−091.37E−09 2.98E−09 2.30E−09 TGI 5.05E−08 1.90E−07 7.69E−09 3.58E−095.60E−09 4.88E−09 LC₅₀ 1.15E−07 1.16E−06 5.92E−08 9.27E−09 1.05E−081.03E−08 63 64 65 66 67 68 A549 GI₅₀ 2.40E−08 5.19E−09 2.29E−08 7.62E−093.27E−08 2.81E−08 TGI 6.14E−08 3.66E−08 5.60E−08 3.97E−08 6.87E−081.13E−07 LC₅₀ 2.73E−07 1.12E−06 3.13E−07 8.74E−07 7.97E−07 6.29E−06 HT29GI₅₀ 9.17E−09 3.58E−09 4.51E−09 5.86E−09 3.79E−08 6.66E−09 TGI 3.27E−071.32E−08 1.96E−07 1.81E−07 1.02E−07 1.50E−08 LC₅₀ 1.35E−05 1.30E−051.32E−05 1.32E−05 1.19E−05 1.42E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀5.64E−09 2.65E−09 3.78E−09 5.34E−09 2.04E−08 5.45E−09 TGI 1.70E−086.24E−09 1.08E−08 1.89E−08 4.53E−08 1.57E−08 LC₅₀ 5.26E−08 2.05E−084.20E−08 7.45E−08 1.00E−07 5.21E−08 OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGILC₅₀ DU145 GI₅₀ 3.48E−09 2.89E−09 2.63E−09 3.39E−09 3.59E−08 4.68E−09TGI 7.24E−09 1.53E−07 6.45E−09 1.60E−09 7.54E−08 1.50E−09 LC₅₀ 4.40E−061.30E−05 4.77E−06 3.86E−06 3.50E−06 1.42E−05 MCF GI₅₀ TGI LC₅₀ MB231GI₅₀ TGI LC₅₀ H-MEC-1 GI₅₀ TGI LC₅₀ LNCAP GI₅₀ 2.86E−09 1.02E−098.75E−10 1.99E−09 3.00E−09 1.59E−09 TGI 5.36E−09 2.67E−09 2.54E−093.83E−09 9.00E−09 3.70E−09 LC₅₀ 1.00E−08 6.12E−09 6.16E−09 7.39E−093.74E−08 8.64E−09 SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ 5.40E−09 1.78E−094.07E−09 2.99E−09 3.12E−08 3.94E−09 TGI 1.50E−08 4.41E−09 1.14E−086.76E−09 7.86E−08 8.01E−09 LC₅₀ 4.49E−09 1.09E−08 2.99E−06 1.60E−076.85E−07 3.51E−08 IGROV-ET GI₅₀ 7.59E−08 3.48E−08 7.60E−08 3.83E−082.68E−08 2.90E−07 TGI 5.37E−07 1.71E−07 4.06E−07 1.79E−07 6.27E−081.66E−06 LC₅₀ 1.33E−05 8.30E−06 3.30E−06 5.59E−06 2.68E−06 8.63E−06SK-BR3 GI₅₀ 3.93E−09 7.17E−10 2.97E−09 6.50E−09 8.71E−09 7.20E−09 TGI8.98E−09 1.50E−07 7.66E−09 3.31E−07 3.74E−08 1.53E−07 LC₅₀ 1.98E−071.30E−05 2.09E−07 1.32E−05 2.84E−07 1.42E−05 K562 GI₅₀ 3.86E−09 8.60E−101.22E−09 4.33E−09 3.39E−08 2.65E−09 TGI 7.92E−09 4.71E−09 1.39E−081.25E−08 3.97E−08 6.60E−09 LC₅₀ 6.34E−07 8.12E−07 7.80E−06 4.84E−072.09E−06 2.34E−07 PANC-1 GI₅₀ 4.60E−09 2.46E−09 3.99E−09 4.57E−092.74E−08 3.90E−09 TGI 1.74E−08 1.09E−08 1.23E−08 2.34E−08 9.27E−081.28E−08 LC₅₀ 4.27E−06 1.30E−05 4.25E−06 6.40E−06 2.78E−06 2.07E−06 LOVOGI₅₀ 8.63E−09 4.15E−09 6.78E−09 5.27E−09 2.86E−08 1.20E−08 TGI 5.48E−081.03E−08 3.69E−08 2.09E−08 7.30E−08 4.80E−08 LC₅₀ 1.35E−05 1.25E−052.96E−06 4.35E−06 1.06E−05 1.93E−06 LOVO-DOX GI₅₀ 3.98E−07 3.20E−073.99E−07 3.70E−07 3.68E−07 4.72E−07 TGI 1.09E−06 2.76E−06 1.05E−061.89E−06 9.75E−07 1.42E−05 LC₅₀ 1.35E−05 1.30E−05 1.32E−05 1.32E−051.27E−05 1.42E−05 HELA GI₅₀ 3.16E−09 2.40E−09 2.34E−09 2.41E−09 3.02E−084.00E−09 TGI 6.29E−09 4.89E−09 4.82E−09 4.68E−09 6.74E−08 7.06E−09 LC₅₀1.25E−08 1.00E−08 9.99E−09 9.07E−09 3.95E−07 1.24E−08 HELA-APL GI₅₀1.10E−08 2.39E−09 2.00E−09 3.05E−09 2.97E−08 3.81E−09 TGI 3.98E−084.80E−09 4.10E−09 6.24E−09 6.47E−08 6.32E−09 LC₅₀ 1.50E−07 9.63E−098.46E−09 1.27E−08 3.27E−07 1.04E−08 69 70 71 72 73 74 A549 GI₅₀ 5.57E−065.06E−06 2.11E−06 5.19E−08 3.25E−06 2.64E−06 TGI 2.23E−05 5.06E−051.05E−05 1.04E−07 6.05E−06 7.09E−06 LC₅₀ 5.57E−05 5.06E−05 3.16E−054.15E−06 1.13E−05 1.61E−05 HT29 GI₅₀ 1.11E−06 1.01E−05 5.27E−06 1.04E−065.65E−06 5.32E−07 TGI 1.11E−06 1.01E−05 5.27E−06 1.04E−06 1.23E−051.61E−06 LC₅₀ 1.11E−05 5.06E−05 5.27E−05 5.19E−06 1.23E−05 1.61E−05SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ TGI LC₅₀ OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀TGI LC₅₀ DU145 GI₅₀ TGI LC₅₀ MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGI LC₅₀H-MEC-1 GI₅₀ 6.65E−06 2.64E−07 TGI 1.23E−05 4.33E−06 LC₅₀ 1.23E−051.61E−05 LNCAP GI₅₀ TGI LC₅₀ SK-OV3 GI₅₀ TGI LC₅₀ IGROV GI₅₀ TGI LC₅₀IGROV-ET GI₅₀ TGI LC₅₀ SK-BR3 GI₅₀ TGI LC₅₀ K562 GI₅₀ TGI LC₅₀ PANC-1GI₅₀ TGI LC₅₀ LOVO GI₅₀ TGI LC₅₀ LOVO-DOX GI₅₀ TGI LC₅₀ HELA GI₅₀ TGILC₅₀ HELA-APL GI₅₀ TGI LC₅₀ 75 76 77 ET729 ET594 ET770 A549 GI₅₀8.89E−07 4.13E−07 3.01E−07 1.82E−08 4.90E−08 3.24E−09 TGI 4.01E−068.40E−07 7.28E−07 5.71E−08 1.31E−07 6.98E−09 LC₅₀ 1.27E−05 4.65E−063.65E−06 1.34E−06 6.54E−07 5.20E−06 HT29 GI₅₀ 3.30E−07 6.27E−07 1.89E−072.05E−09 4.90E−08 5.86E−09 TGI 9.33E−07 1.30E−06 9.08E−07 1.91E−089.80E−08 1.30E−08 LC₅₀ 1.61E−05 1.30E−05 1.31E−05 1.34E−05 1.63E−061.30E−05 SW-620 GI₅₀ 3.27E−08 TGI 9.80E−08 LC₅₀ 3.27E−06 MEL-28 GI₅₀2.38E−07 6.31E−08 7.52E−10 3.27E−08 2.63E−09 TGI 5.49E−07 1.75E−072.11E−09 6.54E−08 5.34E−09 LC₅₀ 1.27E−06 7.56E−07 7.07E−09 1.31E−071.08E−08 OVCAR GI₅₀ 3.27E−08 TGI 1.14E−07 LC₅₀ 1.63E−05 A498 GI₅₀8.17E−08 TGI 3.27E−07 LC₅₀ 1.14E−05 DU145 GI₅₀ 4.92E−07 3.01E−075.68E−10 3.27E−08 4.16E−09 TGI 1.34E−06 1.16E−06 3.17E−09 8.17E−083.20E−08 LC₅₀ 1.30E−05 1.31E−05 1.34E−05 1.63E−07 1.30E−05 MCF GI₅₀3.27E−08 TGI 9.80E−08 LC₅₀ 3.27E−06 MB231 GI₅₀ 3.27E−08 TGI 1.14E−07LC₅₀ 1.63E−06 H-MEC-1 GI₅₀ 2.79E−07 TGI 1.64E−06 LC₅₀ 1.61E−05 LNCAPGI₅₀ 4.66E−08 4.35E−08 3.69E−10 1.48E−09 TGI 8.07E−08 7.10E−08 8.68E−103.06E−09 LC₅₀ 2.57E−07 1.18E−07 3.77E−09 6.34E−09 SK-OV3 GI₅₀ 5.47E−074.39E−07 TGI 1.90E−06 4.86E−06 LC₅₀ 1.30E−05 1.31E−05 IGROV GI₅₀1.97E−07 5.80E−08 4.13E−10 3.32E−09 TGI 6.48E−07 2.72E−07 9.86E−108.51E−09 LC₅₀ 5.25E−06 6.32E−06 7.45E−09 1.30E−05 IGROV-ET GI₅₀ 5.13E−076.52E−07 2.54E−08 1.21E−08 TGI 9.24E−07 1.70E−06 6.19E−08 4.50E−06 LC₅₀1.30E−06 1.31E−05 1.34E−05 1.30E−05 SK-BR3 GI₅₀ 3.98E−40 9.86E−10 TGI8.60E−10 9.48E−08 LC₅₀ 7.11E−09 1.30E−05 K562 GI₅₀ 2.69E−08 1.66E−085.99E−10 1.65E−09 TGI 1.30E−07 1.01E−07 2.97E−09 8.02E−09 LC₅₀ 1.30E−051.31E−05 2.74E−06 6.63E−06 PANC-1 GI₅₀ 6.95E−07 4.18E−07 1.05E−095.53E−09 TGI 4.97E−06 2.72E−06 5.32E−09 1.15E−07 LC₅₀ 1.30E−05 1.31E−051.34E−06 1.30E−05 LOVO GI₅₀ 2.74E−07 2.41E−07 2.01E−09 3.09E−09 TGI6.83E−07 9.17E−07 6.50E−09 5.93E−09 LC₅₀ 1.30E−06 1.31E−05 1.34E−051.14E−08 LOVO-DOX GI₅₀ 2.66E−06 9.29E−07 2.05E−07 7.41E−08 TGI 8.13E−065.66E−06 6.98E−07 1.30E−05 LC₅₀ 1.30E−05 1.31E−05 1.34E−05 1.30E−05 HELAGI₅₀ 3.33E−10 5.09E−09 TGI 6.75E−10 1.26E−08 LC₅₀ 1.78E−09 1.86E−07HELA-APL GI₅₀ 3.41E−10 2.96E−09 TGI 6.79E−10 6.01E−09 LC₅₀ 1.58E−091.22E−08 ET637- ET594- ET736- ET743 ET745 ET759B quinone quinone quinoneA549 GI₅₀ 2.52E−09 7.43E−07 2.62E−08 4.42E−09 2.35E−06 2.56E−08 TGI5.51E−09 4.77E−06 6.13E−08 1.09E−08 4.91E−06 5.29E−08 LC₅₀ 1.21E−081.34E−05 7.02E−07 5.15E−08 1.03E−05 1.09E−07 HT29 GI₅₀ 3.41E−09 5.22E−074.19E−08 3.32E−09 6.53E−07 5.12E−08 TGI 1.11E−08 2.65E−06 2.29E−067.78E−09 1.71E−06 1.28E−07 LC₅₀ 1.31E−05 1.34E−05 1.29E−05 2.26E−061.63E−05 1.30E−05 SW-620 GI₅₀ TGI LC₅₀ MEL-28 GI₅₀ 7.42E−10 6.02E−071.97E−08 2.96E−09 4.13E−07 1.23E−07 TGI 3.06E−09 2.21E−06 4.91E−085.34E−09 7.38E−07 3.00E−07 LC₅₀ 1.12E−08 8.62E−06 1.23E−07 9.61E−091.32E−06 7.18E−07 OVCAR GI₅₀ TGI LC₅₀ A498 GI₅₀ TGI LC₅₀ DU145 GI₅₀6.18E−10 4.88E−07 3.91E−07 3.74E−09 1.99E−06 4.51E−08 TGI 1.29E−092.67E−06 3.20E−06 6.30E−09 5.39E−06 7.63E−08 LC₅₀ 4.33E−06 1.34E−051.19E−05 1.53E−08 1.46E−05 1.29E−07 MCF GI₅₀ TGI LC₅₀ MB231 GI₅₀ TGILC₅₀ H-MEC-1 GI₅₀ 9.03E−09 6.34E−07 TGI 6.88E−08 3.67E−06 LC₅₀ 1.31E−051.34E−05 LNCAP GI₅₀ 2.30E−10 6.52E−07 4.64E−09 1.78E−09 2.74E−072.48E−08 TGI 5.29E−10 2.39E−06 1.67E−08 3.64E−09 4.98E−07 4.46E−08 LC₅₀1.22E−09 9.81E−06 5.22E−08 7.44E−09 9.01E−07 7.99E−08 SK-OV3 GI₅₀1.31E−09 1.32E−06 TGI 1.30E−08 5.48E−06 LC₅₀ 1.31E−05 1.34E−05 IGROVGI₅₀ 5.79E−10 7.74E−07 2.29E−08 2.76E−09 4.52E−07 6.71E−08 TGI 2.60E−093.26E−06 6.52E−08 5.80E−09 9.58E−07 2.15E−07 LC₅₀ 1.03E−08 1.34E−051.00E−05 1.21E−08 3.64E−06 7.35E−07 IGROV-ET GI₅₀ 4.29E−09 3.70E−065.82E−08 3.42E−08 4.37E−06 5.10E−07 TGI 7.86E−09 8.45E−06 2.73E−076.91E−08 9.11E−06 7.82E−07 LC₅₀ 1.86E−08 1.34E−05 1.71E−06 1.39E−071.63E−05 1.20E−06 SK-BR3 GI₅₀ 4.79E−10 6.29E−07 5.37E−09 7.58E−403.02E−07 5.33E−09 TGI 1.72E−09 3.15E−06 1.16E−08 2.67E−09 5.93E−071.13E−08 LC₅₀ 8.57E−09 1.34E−05 8.81E−08 8.51E−09 1.16E−06 1.92E−07 K562GI₅₀ 3.47E−10 8.84E−07 6.00E−09 5.66E−40 1.37E−07 4.96E−09 TGI 6.05E−103.90E−06 3.79E−08 1.97E−09 4.78E−07 1.37E−08 LC₅₀ 1.06E−09 1.34E−051.29E−05 1.44E−08 1.62E−06 1.05E−07 PANC-1 GI₅₀ 1.59E−09 1.32E−063.55E−08 3.72E−09 1.58E−06 3.94E−07 TGI 6.34E−09 9.22E−06 1.22E−078.24E−09 4.86E−06 9.57E−07 LC₅₀ 3.99E−08 1.34E−05 1.10E−05 3.42E−091.49E−05 1.30E−05 LOVO GI₅₀ 1.56E−09 5.55E−06 3.27E−07 3.98E−09 7.59E−072.65E−07 TGI 4.23E−09 1.34E−05 6.21E−08 8.51E−09 2.91E−06 5.96E−07 LC₅₀1.14E−08 1.34E−05 1.18E−07 6.08E−06 1.63E−05 1.30E−06 LOVO-DOX GI₅₀2.95E−08 6.25E−06 1.92E−07 2.62E−07 6.27E−06 7.47E−07 TGI 1.10E−071.34E−05 6.25E−07 9.62E−07 1.31E−05 3.28E−06 LC₅₀ 1.31E−05 1.34E−051.29E−05 9.00E−08 1.63E−05 1.30E−05 HELA GI₅₀ 2.13E−08 2.24E−09 3.43E−073.32E−06 TGI 6.42E−08 4.88E−09 6.20E−07 6.14E−06 LC₅₀ 5.27E−07 1.06E−081.12E−06 1.14E−07 HELA-APL GI₅₀ 3.59E−08 2.39E−09 3.98E−07 5.55E−08 TGI1.02E−07 5.12E−09 6.74E−07 7.84E−08 LC₅₀ 1.04E−06 1.09E−08 1.14E−061.11E−07

1. An ecteinascidin compound having a quinone group in ring E.
 2. Acompound according to claim 1, with a 1,4 bridge.
 3. A compoundaccording to claim 1, with a 1,4-spiroamine bridge.
 4. A compoundaccording to claim 2, wherein the 1,4 bridge is of the formula:—⁽¹⁾CH₂—X—C(═O)—C(R^(a))(R^(b))—S⁽⁴⁾—where the —CH₂— is at the1-position and the —S— is at the 4-position, of the ecteinascidincompound; X is —O— or an isostere; R^(a) and R^(b) together with thecarbon to which they are attached form a group: —C(═O)—; —CHNH₂ or aprotected or derivatised version of such a group; —CHOH or a protectedor derivatised version of such a group; a group of formula:

where R¹ is preferably chosen from hydrogen or substituted orunsubstituted hydrocarbyl, hydrocarbyloxy or hydrocarboyl, and X¹ ishydrogen or substituted or unsubstituted hydrocarbyl, hydrocarbyloxy orhydrocarboyl, or a protecting group; a group of formula:

where R^(d) and X¹ are as defined.
 5. A compound according to claim 1,which is of the formula (Ab):

where R^(a), R^(b), and X are as defined; R⁵ is —OH or a protected orderivatised version of such a group R7 is —OCH3 and R8 is —OH or R7 andR8 together form a group —O—CH2—O—; R^(12b) is a protecting group,hydrogen, substituted or unsubstituted hydrocarbyl, acyl, carbonate,carbamate, cyclic amide or amino acid; and R²¹ is —H, —OH or —CN.
 6. Acompound according to claim 5, wherein R^(a) and R^(b) together with thecarbon to which they are attached form a group: —CO— —CHNX¹X² where X¹is as defined and X² is as defined for X¹; —CHOX¹ where X¹ is asdefined; a group of formula:

where R^(d) is hydrogen or substituted or unsubstituted hydrocarbyl, andX¹ is hydrogen, substituted or unsubstituted hydrocarbylor —(CO)OR′where R′ is substituted or unsubstituted hydrocarbyl; or a group offormula:

where R^(d) and X¹ are as defined.
 7. A compound according to claim 5,wherein R⁵ is —OH or a substituted or unsubstituted acyl derivative. 8.A compound according to claim 5, wherein R⁷ and R⁸ together form a group—O—CH₂—O—.
 9. A compound according to claim 5, wherein R^(12b) ishydrogen, or substituted or unsubstituted hydrocarbyl, amino acid oracyl.
 10. A compound according to claim 5, wherein R²¹ is —OH or —CN.11. A compound according to claim 5, wherein X is —NH— or —O—.
 12. Aprocess which comprises providing as starting material an ecteinascidinwith a substitutent at the N-12 position, and removing that substituent.13. A process according to claim 12, wherein the N—H group isderivatised.
 14. A process for preparing an ecteinascidin compoundwherein a 1, 4 bridge is formed using a 1-labile, 10-hydroxy,12-protected, 18-protected hydroxy, di-6,8-enone fused ring precursorcompound.
 15. A process for preparing an ecteinascidin compound, whereina compound of formula (B):

where R²¹ is as defined, and Prot³ is a protecting group, is subjectedto converting —CH₂NH₂ at the 1-position to —CH₂0H, protecting the —CH₂0Hat the 1-position, protecting the —OH at the 18-position, removing themethyl group at the 12-position, deprotecting the 5-position, forming adi-6,8-enone ring for ring A, protecting the 12-position, removing theprotecting group at the 1-position, forming a labile group at the1-position set up for giving a 1,4 bridge, forming the 1,4 bridge,deprotecting the 18-position, optionally modifying the 1,4 bridge,removing the protecting group at the 12-position, and optionally furthermodifying the structure.
 16. A process according to claim 3, wherein thefurther modifications include one or more of altering the 1-substituent,altering the 5-substituent, altering the 18-substituent, and convertingring E to a quinone.
 17. An ecteinascidin compound which is of theformula (A):

wherein: R^(a) and R^(b) together with the carbon to which they areattached form a group —C(═O)—; a group —CH(R^(c))— where R^(c) is OX¹ orN(X¹)₂ where the each X¹ is H, R′, —C (═O)R′, —O(C═O)R′, substituted orunsubstituted hydrocarbyl; or a spiro ring; R⁵ is —OH or a protected orderivatised version of such a group; R⁷ is —OCH₃ and R⁸ is —OH or R⁷ andR⁸ together form a group —O—CH₂O—; R¹² is a protecting group; R²¹ is —H,—OH or —CN; X is —NH— or —O—; ring E is of the formula:

where R¹⁸ is —OH or a protected or derivatised version of such a group.18. A process for preparing an ecteinascidin compound, in which theprotecting group on N-12 in a compound of claim 5 is removed to give ahydrogen.
 19. A process according to claim 18, in which the hydrogen onN-12 is replaced with another substituent R^(12a).
 20. An ecteinascidincompound of the formula (Ac):

where R^(a), R^(b), R⁵, R⁷, R⁸, R^(12b), R²¹, and ring E are as definedin claim 17.